Tall South African Kevin Anderson smacked 22 aces in dousing the second-seeded Wawrinka 7-6 (7-4), 7-6 (13-11) at the prestigious Queen's Club.
The hard-hitting Swiss star Wawrinka has captured two of the last six Grand Slam events.
In other action involving seeds in London, No. 3 Canadian Milos Raonic crushed 29 aces in getting past Frenchman Richard Gasquet 6-4, 6-7 (5-7), 6-1 and No. 7 Frenchman Gilles Simon grounded Aussie wild card Thanasi Kokkinakis 6-4, 6-2.
Raonic will face Simon in Friday's quarterfinals.
Second-round matches will come Thursday for top-seeded Andy Murray, fourth-seeded U.S. Open champ Marin Cilic, sixth-seeded reigning Queen's Club titlist Grigor Dimitrov and eighth-seeded Spaniard Feliciano Lopez, who will take on 6-foot-10 American John Isner.
The 2015 Aussie Open runner-up and former Wimbledon titlist Murray is a three-time Queen's Club winner, while Cilic titled here in 2012 and was the runner-up to Murray in 2013.
The Bulgarian Dimitrov beat Lopez in last year's Queen's Club finale.