This study aimed to investigate the effects of leucine (Leu) on the synthesis and secretion of digestive enzymes in cultured pancreatic tissue of dairy goats and on the signaling molecules. Fresh pancreatic tissue from dairy goats was cut into approximately 2 mm × 2 mm pieces and incubated in oxygenated Krebs-Ringer bicarbonate buffer containing 0 (the control), 0.40, 0.80, or 1.60 mM Leu at 39°C in a CO2 incubator for 180 min. The results showed that Leu increased the release of α-amylase, trypsin, and chymotrypsin in the buffer and tissue, as well as the total activity (P < 0.05), especially at 0.40 and 0.80 mM. Compared with the control, 1.60 mM Leu increased the release of α-amylase and the total activity of trypsin and chymotrypsin (P < 0.05) but had no effect on the tissue concentration of α-amylase, trypsin, and chymotrypsin or the total activity of α-amylase (P > 0.05). Leu improved the mRNA expression of α-amylase, trypsin, and chymotrypsin (P < 0.05), especially at 0.80 and 1.60 mM. The activity and mRNA expression of lipase were not affected (P>0.05). Compared with the control, 0.40 and 0.80 mM Leu increased the expression of the γ isoform of4EBP1 (P < 0.05), implying increased phosphorylation of4EBP1. Leu increased the phosphorylation of S6K1 (P<0.05). Compared with the control, 0.40 and 0.80 mM Leu decreased the eEF2 phosphorylation level (P<0.05). Conclusively, these results suggested that Leu could regulate the synthesis of pancreatic enzymes by increasing the mRNA expression and phosphorylation level of protein factors in the mammalian target of rapamycin pathway and the optimal Leu level in this experiment was 0.80 mM.