This study was investigated the effects of dietary supplementation of leucine and phenylalanine on the development of the gastrointestinal tract and the intestinal digestive enzyme activity in male Holstein dairy calves. Twenty calves with a body weight of 38 + − 3 kg at 1 day of age were randomly divided into four groups: a control group, a leucine group (1.435 g · l −1 ), a phenylalanine group (0.725 g · l −1 ), and a mixed amino acid group (1.435 g · l −1 leucine plus 0.725 g · l −1 phenylalanine). The supplementation of leucine decreased the short-circuit current (Isc) of the rumen and duodenum (P<0.01); phenylalanine did not show any influence on the Isc of rumen and duodenum (P>0.05), and also counteracted the Isc reduction caused by leucine. Leucine increased the trypsin activity at the 20% relative site of the small intestine (P<0.05). There was no difference in the activity of α-amylase and of lactase in the small intestinal chyme among four treatments (P>0.05). The trypsin activity in the anterior segment of the small intestine was higher than other segments, whereas the α-amylase activity in the posterior segment of the small intestine was higher than other segments. Leucine can reduce Isc of the rumen and duodenum, improve the development of the gastrointestinal tract, and enhance trypsin activity; phenylalanine could inhibit the effect of leucine in promoting intestinal development.