Thirty-two Chinese Holstein heifers (98 d old) were randomly divided into 4 groups with 8 heifers each (8 replications). Each group was subjected to one of the following 4 total mixed rations (TMR) with 2.52 (A), 2.82 (B), 2.99 (C), and 3.21 (D) Mcal of metabolic energy (ME) per kg (dry matter basis) diet for 82 d. The energy gradient was adjusted by changing the composition of concentrate and adding rumen bypass fat. The concentrate to roughage ratio of the TMR was fixed at 59.52:40.48. Growth performance was determined at 98, 120, 150, and 180 d of age, and rumen fermentation parameters were determined at 98, 120, 150, and 180 d of age, and microbial populations in rumen fluid from 181-d-old heifers were determined by real time polymerase chain reaction analysis. During 98 to 180 d of age, the average daily gain (ADG) did not differ among the groups (P > 0.05). However, during 151 to 180 d of age, ADG of the heifers of group D (1.11 kg/d) were greater (P < 0.05) than those of group A (0.77 kg/d). Feed utilization efficiency was improved in groups C and D (P < 0.05) when compared with that in group A. Dietary ME did not influence ruminal pH value and NH₃-N concentration (P > 0.05). Increasing level of ME significantly increased ruminal proportion of propionate (P < 0.01) and reduced that of acetate (P < 0.05) and the ratio of acetate to propionate (P < 0.01). Dietary treatments had no significant effect on the concentration of total volatile fatty acid (P > 0.05). Lachospira multipara of group C tended to be higher than that of group D (P < 0.10), and the numerical quantity of protozoan, R. flaefaciens, R. albus, B. fibrisolvens and P. ruminicola of group C were greater than the other groups (P < 0.05). Current study suggested that heifers supplemented with 2.99 Mcal of ME per kg of diet (group C) had better feed utilization efficiency and more abundant microbial flora compared with those fed other levels of ME.