In ruminants, urea was broken down rapidly to ammonia by rumen bacteria and urease inhibitors were used for increasing the efficiency of urea utilization by inhibiting ruminal urease. However, the effect of urea and urease inhibitors on the rumen microbes was not clear. This study investigated the effect of urea and AHA (acetohydroxamic acid) addition in the diets on rumen microbial diversity using dual-flow continuous culture systems. Eight fermenters were used in a period of 10 d (7 d for adaptation and 3 d for sampling) experiment and TMR (containing alfalfa hay 17.72%, corn silage 17.5%, steam corn 7.39%, soybean meal 2.64%) were placed into each fermenter twice a day. Based on this diet, the fermenters were assigned to a 2 × 2 factorial arrangement of treatments with urea supplemented at 0 or 0.5% dry matter intake (DMI), and AHA equivalent to 0 or 450 mg/kg DMI. While the urea and AHA were dissolve in the artificial saliva and infused into the vessels twice daily. On each sampling day, fermentation fluids were collected at 0 h, 2 h, 6 h and 10 h from each fermenter. Total DNA of rumen microbe were extracted and subjected for DGGE and 16S rRNA gene sequencing. Distinct bacterial profiles were observed with urea addition and little differences were found with AHA addition. UPGMA analysis showed that samples with urea and AHA addition were clustered together. Group with urea addition showed a higher Shannon diversity compared with other groups (P < 0.01). 16S rRNA gene sequencing analysis revealed that the dominant ruminal bacteria shared by all 4 groups belonged to phyla Firmicutes, Bacteroidetes and Proteobacteria. However, in urea adding groups, the bacteria Lachnospiraceae, Clostridiaceae, and Succinivibrionaceae were found in highest abundance compared with the other 2 groups (P < 0.01). In contrast, the Paraprevotellaceae and Veillonellaceae bacteria were abundant in treatments without urea (P < 0.01). Little difference of the bacteria abundance was found with AHA addition. In conclusion, adding urea to the diet could change the ruminal bacteria diversity while AHA addition had little effect on the rumen microbiota.