AbstractDiet 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. The impact of TR feeding on the GM composition has been mostly explored by means of metagenomic sequencing. To date, however, little is known about the modulation of GM functions by this dietary regimen. Here, we analyzed the effects of TR feeding on GM functions by evaluating protein expression changes in a rat model through a metaproteomic approach. We observed that TR feeding has a relevant impact on GM functions, specifically leading to an increased abundance of several enzymes involved in carbohydrate and protein metabolism and expressed by Lactobacillus spp. and Akkermansia muciniphila. Taken together, these results contribute to deepening our knowledge about the key relationship between diet, GM, and health.