The objective of this study was to evaluate the effect of different levels of dietary crude protein (CP) on mRNA expressions of urea transporter B (UT-B) and aquaporins (AQP) and nitrogen metabolism in liver and rumen epithelium of 5-mo old Holstein calves. Ten intact Holstein calves (bull, 172.80 ± 27.73 kg BW) were randomly assigned to 2 treatments (n = 5 calves/treatment): 1. complete nutritional palletized feeds containing 21.02% of CP (HP); 2. complete nutritional palletized feeds containing 15.05% of CP (LP). All 10 bull calves were slaughtered at the end of 35-d trial period. Blood samples were collected at 0 h, 1 h, 3 h and 5 h after feeding at the day before slaughter for analyzing hematological parameters. After slaughter, rumen contents were obtained for ammonia nitrogen and urea nitrogen analysis. Meanwhile, liver and rumen epithelial tissue was collected immediately and the mRNA expression of UT-B and AQP was detected by quantitative PCR. Data were analyzed by the t-test with statistical significance indicated by P < 0.05 (SPSS 19.0). Our results showed that the concentrations of rumen ammonia nitrogen (P = 0.001) and urea nitrogen (P = 0.001) were both significantly higher in HP group compared with LP group. The concentrations of blood urea nitrogen (BUN) were higher in HP group at 1 h (P = 0.005), 3 h (P = 0.012) and 5 h (P = 0.016) after feeding. In comparison with LP group, the relative mRNA expression of AQP-3 in liver of HP group was significantly increased (P = 0.011), but there was no significant difference in the relative mRNA expression of UT-B (P = 0.602) and AQP-10 (P = 0.135). Similarly, there was no significant difference in the relative mRNA expression of UT-B (P = 0.862) and AQP-3 (P = 0.706) in rumen epithelium between 2 groups. In conclusion, high dietary CP level increased the mRNA expression of AQP-3 in liver of bull calves, which might indicate that AQP-3 may be an important transporter of hepatic nitrogen metabolism and therefore it is candidate to be further studied in hepatic nitrogen metabolism experiments.