Diet is a key factor influencing gut microbiota (GM) composition and functions, which in turn affect host health. Among dietary regimens, time-restricted (TR) feeding has been associated to numerous health benefits. To date, little is known about the modulation of GM protein functions by TR feeding. Here, we analyzed the effects of TR feeding on GM protein expression in a rat model through a metaproteomic approach. We observed that TR feeding has a relevant impact on GM functions, especially on several biological activities exerted by members of the genus Lactobacillus, including proteolysis and metabolism of galactose-containing glycans.