The objective of this experiment was to investigate the effects of urea and acetohydroxamic acid (AHA) on in vitro ruminal urea-nitrogen (urea-N) metabolism and microbial community using total mixed ration as a substrate. Treatments were arranged in a 2 × 2 factorial design with urea supplemented at 0 or 2% dry matter (DM), and AHA equivalent to 0 or 450 mg/kg DM. Ruminal fluids were collected from 3 Chinese Holstein dairy cows through permanent ruminal fistula, diluted with artificial saliva (1: 2, v/v), and incubated anaerobically at 39°C for 0, 1, 2, 4, 6, and 12 h. Each treatment was performed in 3 serum bottles and experiment was run 3 times. Supplementation of urea increased (P < 0.01) ruminal pH, ammonia-nitrogen (NH₃-N) concentration and urease activity, while addition of AHA inhibited (P < 0.01) their increments. Acetohydroxamic acid was still stable within 6 h of incubation. When AHA was added, urea-N concentration of fermentation fluid in the treatment with urea supplied was gradual decline (P < 0.05). The peak of NH₃-N concentration was not delayed by AHA addition, comparing with treatment with urea supplementation only. The bacterial PCR-DGGE profiles of 4 treatments were similar to each other before and after incubation. Urea stimulated (P < 0.01) the decrements of Ruminococcus albus, R. flavefaciens, Fibrobacter succinogene, and Butyrivibrio fibrisolvens populations, but had no effect on Prevotella population (P = 0.18). However, all those functional bacterial populations were not influenced by AHA addition. It was concluded that AHA could slow down the degradation of urea by the inhibition of urease activity, and the dose of 450 mg/kg DM could not alter ammonia formation pattern in this fermentation condition. In addition, AHA had no effect on ruminal microbiota, which could be altered by urea supplementation.