Russian was the only vaulter to clear 4.89 metres, American LeShawn Merritt wins men's 400m final and Germany's Robert Harting wins a third world title in men's discus.
Isinbayeva wins pole vault gold at World Athletics Championships
The Russian, twice Olympic champion and world outdoor record holder, was the only vaulter to clear 4.89 metres.
It was a season-best leap for the vaulter, 31, the first woman to clear the revered five metres barrier, who said before the World Championships that she would retire.
American Olympic champion Jenn Surh took silver and Cuban Yarisley Silva bronze. Both failed at 4.89m after clearing 4.82m.
After a nervous start when she failed with her first attempt after entering the competition at 4.65m, a measure of her confidence with her rivals already up and running, Isinbayeva grew in stature.
With a chorus of "Yelena Yelena" reverberating around the Luzhniki Stadium, the biggest crowd of a poorly attended championships so far, each clearance was greeted by a cacophony of approval.
Isinbayeva rewarded the support with punches in the air and squeals of delight. When gold was assured she ran to the crowd and embraced her coach.
Milking the moment and urging the crowd to raise the decibel level further, she asked for the bar to be raised to 5.07m, one centimetre above the world record she set in 2009.
Three unsuccessful attempts followed, but it could not spoil the former gymnast's celebrations as she set off for a lap of the track which featured a cartwheel and back-flip.
Isinbayeva was virtually unbeatable between 2003 and 2008, when she kept raising the bar higher and higher, often by a centimetre at a time.
But plagued by injury and poor form and after failing to register a height in the 2009 World Championships, she decided to take a break from the sport, returning after an 11-month absence.
She was again outside the medals at the 2011 worlds, but took bronze at last year's London Olympics.
LaShawn Merritt returned the United States to the top of the 400m podium with a commanding performance as defending champion Kirani James faded badly to finish seventh.
Merritt took the race on from the gun and went out hard over the first 200m, but unlike James, the American had plenty left in the tank coming into the home straight to seal victory in a world-leading 43.74 seconds.
Compatriot Tony McQuay was second in 44.40 and fast-finishing Luguelin Santos of Dominican Republic edged out Belgium's Jonathan Borlee for bronze in 44.52.
Merritt was world champion in 2009, but finished behind the young Grenadian James in Daegu two years ago. He is the only man to beat James this season.
"It just didn't work out for me today," said James, 20, who beat Merritt for gold two years ago.
"It is back to the drawing board, see what's going on. I didn't come into the worlds expecting this but you just move forward from this."
German discus great Robert Harting won his third successive title at the World Championships in Moscow on Tuesday.
The Olympic champion, 28, threw a best of 69.11 metres to take gold, while Piotr Malachowski of Poland was second at 68.36m and Gerd Kanter of Estonia took bronze with a toss of 65.19m.
Ukraine's Ganna Melnichenko deprived Mr and Mrs Ashton Eaton of a famous multi-event double by claiming gold in the heptathlon at the World Championships on Tuesday.
The gruelling two-day event came down to the seventh and final event, the 800 metres, with Canada's Brianne Theisen Eaton, wife of newly crowned world decathlon champion Ashton Eaton of the United States, handed a mighty task.
The Canadian had to beat the Ukraine athlete by a margin of 4.69 seconds to claim gold at a packed Luzhniki Stadium. It was not to be, however, as Melnichenko tracked Theisen Eaton around the strength-sapping 800m after the latter had shot to the front, tying up badly late in the race.
Melnichenko's time of two minutes, 9.85 seconds was enough for her to claim victory in the keenly contested heptathlon that offered a great cut-and-thrust spectacle over two days.
Melnichenko amassed a total of 6,586 points, a personal best, while Theisen Eaton claimed silver with 6,530 points and was comforted by Eaton trackside.
Dafne Schippers of the Netherlands took bronze with a Dutch-record 6,477 points.
The Ethiopian runner Mohammed Aman won the men's 800m title to claim the country's first medal in the two-lap event.
Aman clocked a season-best of 1:43.31, with American Nick Symmonds taking silver in 1:43.55.
Djibouti's Ayanleh Souleiman claimed bronze (1:43.76), the first medal for the east African country since Ahmed Salah's silver in the marathon in the 1991 worlds in Tokyo.
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