The big-serving American, seeded ninth in the Spanish capital, hit 14 aces on his way to a 7-6(8) 6-4 win - his first on clay this season.
“I feel like I competed very well out there and on top of that I felt like I played well from start to finish,” said Isner.
“I played really well in the first set. Had a lot of chances, but I just didn’t capitalise on them. I was able to win a super-close tie-break.
"I served extremely well. When I serve like that, I’m very tough to beat and it bodes well for me.
Number 10 seed Kei Nishikori also won through with a 6-4 6-4 win over Ivan Dodig of Croatia, while 11th seeded Frenchman Jo-Wilfried Tsonga found the going a little tougher in a 6-3 4-6 6-3 victory over compatriot Edouard Roger-Vasselin.
"I started well," said Japan's Nishikori. "I broke his serve in the first game. He's not easy to play. He has a great serve and he comes to the net sometimes.
"But I played really well. There was just one bad game, on my serve at 3-2 in the second set. Otherwise, it was an almost perfect match.”
Tsonga, who took two hours and 10 minutes to beat Roger-Vasselin, said: "It was a difficult match. I played well in the second set, but I lost my concentration a bit and then it was too late to come back. But in the end I won, and that's good."
In the women's draw, Sam Stosur sprung the first upset of the tournament, the Australian beating ninth-seeded Dominika Cibulkova 6-4 6-0.
Although Stosur is ranked lower than Cibulkova, she has now won all four of their meetings, the latest victory coming after she went on a run to claim 12 of the last 13 games.
There were no problems for the top seeds in action on the opening day though, with Li Na, Agnieszka Radwanska and Simona Halep - two, three and four respectively - all winning through in straight sets.
Madrid Masters men's results
12-Grigor Dimitrov (Bulgaria) beat Pablo Carreno (Spain) 6-2 6-4
Santiago Giraldo (Colombia) beat Lleyton Hewitt (Australia) 7-5 4-6 6-2
Feliciano Lopez (Spain) beat Federico Delbonis (Argentina) 7-6(4) 4-6 6-2
Fernando Verdasco (Spain) beat Andreas Seppi (Italy) 6-3 7-6(3)
Gilles Simon (France) beat Benoit Paire (France) 2-0 (Paire retired)
Albert Ramos (Spain) beat Nicolas Mahut (France) 6-1 7-6(7)
15-Mikhail Youzhny (Russia) beat Benjamin Becker (Germany) 6-3 6-4
Marinko Matosevic (Australia) beat Philipp Kohlschreiber (Germany) 6-2 6-2
11-Jo-Wilfried Tsonga (France) beat Edouard Roger-Vasselin (France) 6-3 4-6 6-3
9-John Isner (U.S.) beat Teymuraz Gabashvili (Russia) 7-6(8) 6-4
Dominic Thiem (Austria) beat Dmitry Tursunov (Russia) 6-4 6-2
10-Kei Nishikori (Japan) beat Ivan Dodig (Croatia) 6-4 6-4
Marius Copil (Romania) beat Marcel Granollers (Spain) 6-3 6-4
Jarkko Nieminen (Finland) beat Robin Haase (Netherlands) 6-3 6-2
Madrid Masters women's results
14-Carla Suarez Navarro (Spain) beat Yvonne Meusburger (Austria) 7-6(6) 6-4
6-Jelena Jankovic (Serbia) beat Elina Svitolina (Ukraine) 6-4 6-3
13-Caroline Wozniacki (Denmark) beat Ekaterina Makarova (Russia) 6-2 6-2
4-Simona Halep (Romania) beat Julia Goerges (Germany) 6-2 6-0
Svetlana Kuznetsova (Russia) beat Alize Cornet (France) 6-3 4-6 6-1
15-Sabine Lisicki (Germany) beat Karolina Pliskova (Czech Republic) 6-2 6-7(5) 6-3
3-Agnieszka Radwanska (Poland) beat Eugenie Bouchard (Canada) 7-6(3) 6-2
Anastasia Pavlyuchenkova (Russia) beat Maria Teresa Torro (Spain) 6-4 4-6 6-1
Irina Begu (Romania) beat Zhang Shuai (China) 6-4 7-5
2-Li Na (China) beat Kirsten Flipkens (Belgium) 6-1 7-6(7)
Samantha Stosur (Australia) beat 9-Dominika Cibulkova (Slovakia) 6-4 6-0
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- Kei Nishikori