The American, last year's runner-up, needs to win the tournament if he is to clinch the final berth in the season-ending event at the O2 in London from Nov. 12-19.
Del Potro would have claimed it if he had won, but the ninth seeded Isner was more consistent and set up a meeting with Serbian qualifier Filip Krajinovic, who advanced after world number one Nadal pulled out.
Nadal told a news conference he was suffering from a right knee problem, leaving his participation in the Finals in the balance and robbing fans in Paris of the tournament's prime attraction.
The home support had something to cheer, however, when wildcard Julien Benneteau defeated Croatian third seed Marin Cilic 7-6(5) 7-5 to advance to the last four.
He will face American Jack Sock, who also stayed in contention to secure the last ATP Finals berth with a 6-7(3) 6-2 6-3 win over Spain's Fernando Verdasco.
Sock needed almost 2-1/2 hours and five match points before he finally tamed Verdasco in a late-night thriller that finished at almost 1am local time.
Pablo Carreno Busta is currently in pole position to bag the final spot in the season-ending finale but Isner or Sock would overtake the Spaniard by claiming the Paris title.
While the top seeds have been paying the price of a demanding season, Isner has looked fresh and despite allowing Del Potro to level for one-set all with a double fault on set point, the American was a cut above his opponent.
"I definitely wasn't preparing to play Rafa (in the following round), today, it was all about Del Potro," he said.
"You would think Rafa, if he took the court would be the favourite in that match. But I think it just goes to show how physical tennis is, especially with someone like Rafa who's played a lot this year. I don't know what's going through his head, but I can't control that.
"So whether I was playing Rafa or Filip tomorrow, it's going to be a pretty similar game plan for me. So that's what I'm focussing on."
Benneteau was also the fresher player when he faced Cilic and played the important points far better than his opponent.
Cilic double faulted to hand the opening set to Benneteau and the Frenchman played the closing stages of the second perfectly, wrapping up the match with a superb forehand winner down the line on the Croatian's serve.
(Reporting by Julien Pretot; Editing by Andrew Heavens/Toby Davis/Pritha Sarkar)
