The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.