The purpose of this 42-day study was to examine the effect of dietary vanadium on intestinal microorganism diversity in the duodenum, ileum, cecum, and rectum segments of broilers by the plate count and polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE). A total of 420 1-day-old avian broilers were divided into six groups and fed on a control diet or the same diet supplemented with vanadium at the doses of 5, 15, 30, 45, and 60 mg/kg in the form of ammonium metavanadate. In comparison with control group, the dietary vanadium at the doses of 45 and 60 mg/kg could decrease the counts of Bifidobacterium spp. in the intestinal tract at 21 and 42 days of age. With increasing level in dietary vanadium, the counts of Escherichia coli were significantly increased in the ileum, cecum, and rectum and were decreased in the duodenum at 21 and 42 days of age. However, the counts of Lactobacilli were decreased in the cecum and rectum and increased in the ileum of 45 and 60 mg/kg groups. The colonization of these three bacteria could be affected by dietary vanadium. DGGE analysis showed that the number of bands in duodenum, ileum, cecum, and rectum were obviously decreased in the 30, 45, and 60 mg/kg groups at 21 and 42 days of age. In conclusion, the dietary vanadium in excess of 30 mg/kg could alter the amount and diversity of intestinal bacteria in broilers, implying that the structure and initial balance in the intestinal microbiota were disrupted.