Diet changes the composition and function of the gut microbiota (GM). Short-chain fatty acids (SCFAs) are mainly produced through carbohydrate fermentation by the GM. It remains unclear whether changing the diet influences the accumulation of SCFAs in the grass carp (Ctenopharyngodon idellus) gut. To address this question, we compared SCFA accumulation with GM changes in the hindgut contents of grass carp in groups fed hybrid giant napier (Pennisetum sinese Roxb, HG), formula feed (FG), and broad bean (Vicia faba, BG). Our results showed that diet significantly influenced the SCFA concentrations in the hindgut of grass carp. By eliminating the influences of dietary SCFA, acetate and total SCFA accumulation in the hindgut of grass carp turned out to be positively associated with dietary cellulose contents. Most of the enriched bacterial biomarkers in the HG group that consumed the most dietary cellulose were SCFA-producing bacteria. These results implied that diet could regulate the relative abundances of potential SCFA-producing bacteria and SCFA accumulation in the grass carp hindgut. These findings shed light onto GM functions related to SCFA production and associative functions between SCFAs and the GM of grass carp.