Running the third leg, the 100 and 200m specialist received the baton in first place and handed off without giving up any ground in a 45.68-second split, the third fastest of his team.
Alexander Doom, the gold medallist in the 400 a night earlier, pipped American anchor Christopher Bailey at the finish line to give Belgium gold in three minutes 2.54 seconds. The US crossed in 3:02.60.
Lyles, who led the US team out during introductions with a loud "Woo!", raced to silver in the 60m on Friday in his world indoor debut.
Lyles won the 100m, 200m and 4x100m at last year's world championships in Budapest, becoming the first man since Usain Bolt in 2015 to complete the sprint treble.
He hinted recently he is considering going for four golds at this year's Paris Olympics, adding the 4x400 to his schedule.
"I've never had somebody tell me something that has thrown my out-of-the-box thinking to inside-the-box, but that was like: okay, I'm not going to say no to that," Lyles told L'Equipe recently.
"After what I did at Budapest and seeing what my body can handle, if I train for it, okay, let's take a shot. It's the last race, there's nothing to lose."
Lyles' relay run prompted some trash talk on social media, with American sprinter Fred Kerley suggesting USA Track and Field was showing favouritism by putting Lyles in the relay.
"He could be here, but he ain't. If he mad, come on out here," Lyles told reporters in response.
Lyles is no stranger to the one-lap race. He famously ran a 45-second split as a high schooler at the Penn Relays, making up huge ground to take his team from second last to second.
