To better understand the molecular mechanisms underlying digestion and absorption in the intestine of the darkbarbel catfish (Pelteobagrus vachellii), we studied the intestinal transcriptome between day and night‐time. In this study, two triplicate groups of P. vachellii (1.05 ± 0.36 g) were fed either at 08:00 (D24 group), control) or 20:00 (W24 group) with the same amount of commercial feed. A significant increase of the apparent digestibility (AD) of protein and lipid was observed in the night group (p < 0.05). Comparison of gene expression levels revealed that 1,125 genes were significantly up‐regulated and 364 genes were significantly down‐regulated in the night group. Among them, 54 digestion‐ and absorption‐related genes, apolipoprotein A‐I, trypsin 1b, solute carrier family 26 member 6 b, sodium/glucose cotransporter 1, cholecystokinin B, maltase‐glucoamylase, diacylglycerol O‐acyltransferase 2, ATP‐binding cassette subfamily A member 1‐like and phospholipase A2, etc., were up‐regulated. Additionally, in the night group, 18 carbohydrate metabolism‐related genes, 17 protein metabolism‐related genes and 16 lipid metabolism‐related genes were up‐regulated, including fatty acid‐binding protein, diacylglycerol O‐acyltransferase 2, adipose triglyceride lipase, phospholipase A2, glucose phosphate isomerase, glycogen phosphorylase, arginase‐2, d‐amino‐acid oxidase, glutamate dehydrogenase, glycine n‐acyltransferase, tryptophan 2,3‐dioxygenase, kynureninase, cysteine dioxygenase type 1 and glycine dehydrogenase. The results indicated that a shift in feeding time from day to night may improve the digestion and absorption of proteins and lipids. Moreover, the amino acid metabolism, triglyceride synthesis, gluconeogenesis may be also influenced by the shifted feeding time.