Federer, a four-time winner at the event, beat del Potro 6-3, 7-5 at the Lindner Family Tennis Center.
Del Potro wasn't able to repeat his feat of 2009 when he stunned Federer in the 2009 U.S. Open final.
Also in second-round action Tuesday, eighth-seeded Czech Tomas Berdych advanced with a 7-6 (7-2), 6-0 win over Argentine Juan Monaco and American James Blake, the 2007 Western & Southern runner-up, bested Italian Fabio Fognini 6-4, 6-2.
Other second-winners included ninth-seeded Nicolas Almagro of Spain, 10th-seeded Frenchman Gilles Simon and 15th-seeded countryman Jo-Wilfried Tsonga, as well as French left-hander Michael Llodra, who upset 13th-seeded Russian Mikhail Youzhny.
Celebrity Couples of 2014 [PHOTOS]