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Simple Summary: Oligopeptide transporter 1 (PepT1) plays a role in the transportation and absorption of oligopeptides, which is an important part of protein nutrition and affects the growth of animals. Tributyrin (TB), the precursor of butyrate, exhibits similar functions to those of the butyrate in intestinal nutrients absorption. The analysis of TB on the growth of grass carp and its regulation pathway on PepT1 may help us to better understand the functions of TB and oligopeptide transportation via PepT1, which can be modulated by diet. In this study, we demonstrated that an appropriate level of tributyrin supplementation in the diet promoted the growth of juvenile grass carp and elevated the expressions of caudal type homeobox 2 (CDX2), specificity protein 1 (SP1), and PepT1 in the grass carp intestine and primary intestine cell. In addition, CDX2 and SP1 regulating the expression of PepT1 was investigated. Finally, CDX2/SP1-mediating tributyrin regulation on PepT1 was elucidated. This study verified the effect of tributyrin on the growth of juvenile grass carp and clarified the tributyrin regulation pathway on CDX2/SP1-PepT1. The nutritional functions of tributyrin (TB) have been extensively studied, but questions remain regarding its influence on the growth of juvenile grass carp (Ctenopharyngodon idellus) and the regulation pathway to PepT1 in the intestine of grass carp. To answer the remaining questions, feeding trials, cell trials, and peritoneal injection trials were conducted in this study. The results showed that an appropriate level of TB (0.5 g/kg and 1.0 g/kg) supplementation in feed significantly promoted the growth performance of juvenile grass carp. The expressions of intestine genes (CDX2, SP1 and PepT1) related to oligopeptide transportation increased in the 0.5 g/kg TB group of feeding trials and both the 5 mM and 10 mM TB groups of the intestine cell trials, respectively. Subsequently, the injection trials of inhibitors CDX2 and SP1 demonstrated that the inhibition of CDX2 or SP1 decreased the mRNA expression of PepT1. Finally, the results of independent or combined treatments of TB and the inhibitors suggested that CDX2/SP1 mediated TB regulation on PepT1. These findings may help us to better understand the functions of TB on growth and PepT1 oligopeptide transportation, which could be modulated by dietary TB through the CDX2/SP1-PepT1 pathway in juvenile grass carp. [ABSTRACT FROM AUTHOR]