The effects of a grain source (corn grain (CG) vs. barley grain (BG)) and starter protein content (19% vs. 22% CP, dry matter basis) on growth performance, digestibility, ruminal fermentation, and blood metabolites were evaluated in Holstein dairy calves. Forty 3-day-old female calves with a starting body weight of 39.3 kg were subjected to four treatments in a completely randomized design with two by two factorial arrangements. Treatments were: (1) CG + 19% CP (CG-19CP); (2) CG + 22% CP (CG-22CP); (3) BG + 19% CP (BG-19CP); and (4) BG + 22% CP (BG-22CP). All calves were weaned at 59 days of age and remained in the study until 73 days of age. Starter and total DM intake were not affected by grain source and dietary protein content (p > 0.05). The average daily gain and feed efficiency were improved, and ruminal total short-chain fatty acid, propionate, and butyrate concentrations were increased in BG calves compared to CG calves (p < 0.05). The ruminal concentrations of ammonia nitrogen (d 71; p = 0.02) and acetate (d 35; p = 0.02) were increased in CG fed calves compared to BG. The greatest wither height (p = 0.03) and blood insulin concentration (p = 0.03) were seen in BG-22CP treatment. In conclusion, BG has marginal benefit in the height of calves when fed with diet containing 22% CP which may be recommendable in replacement heifer rearing programs.