Makwala had the men's 400m title race shot to bits with more than 50 metres to run before cruising over the line in 44.35 seconds and celebrating with 10 trademark push-ups.
Kenyan Beatrice Chepkoech made the early running, with Australian Linden Hall on her shoulder.
But it was always a matter of when, rather than if, the controversial South African would make her move.
It happened with 250m to go and after that the race was as good as over.
Semenya went on to win in a Games record time of four minutes 00.71 seconds ahead of Chepkoech and Melissa Courtney from Wales, who went past a brave Hall in the dying stages.
Hall and her fast-finishing compatriot Georgia Griffith were fourth and fifth respectively.
As the world's most famous hyperandrogenous athlete, Semenya is never far from controversy.
But on Tuesday night she was more interested in talking about her quest for an 800-1500m double at the 2018 Games.
"For us it means a lot," she said.
"It shows that we are matured, we are growing. We are still learning how to run middle distance.
"It's difficult to balance speed with endurance, but with 10 years of experience for me now I'm ready for anything.
"I'm more confident that I know how to run in fast races and slow races, to muster the kicks and make sure I'm in the right shape because I can't always run in front."
Makwala has made a habit of doing a series of push-ups after each win - he usually bangs out five for a regular race and 10 for a big win.
"I just want to show that Makwala is still there, that I still have the power to go," he said after leading Baboloki Thebe across the line for a Botswanan 1-2.
"I came here ready for the gold medal.
"I was training hard for this and you can see from my heat and the final I was just relaxing.
"I knew that if I reached 300 metres first, those guys couldn't manage me."
Australian Steve Solomon was never in contention and finished seventh in 45.64.
Makwala has yet to decide whether to attempt the 200-400m double at next year's world championships in Doha or to focus on the one-lap race.
"If my fitness level is good I will go for the double," he said.