To elucidate the characteristics of group B streptococcus (GBS)-positive vaginal flora in pregnant women, vaginal cultures were conducted in 4025 women at 22 to 36 weeks of gestation. The results were analyzed by Fisher's exact test. Among 4025 women, 408 were found to be GBS positive and 3617 were GBS negative (GBS-negative group). A total of 1151 bacterial strains were recovered in the GBS-positive group and 6746 strains in the GBS-negative group. The percentages of Gram-positive cocci other than GBS, anaerobes, fungi, and Lactobacillus were 18.8%, 1.4%, 6.0%, and 34.4%, respectively, in the GBS-positive group, and 30.4%, 4.1%, 8.8%, and 53.5% in the GBS-negative group. The percentages were significantly lower in the GBS-positive group (P < 0.0001, P < 0.0001, P = 0.0012, P < 0.0001, respectively). Judging from the reduction in Lactobacillus, the GBS-positive vaginal flora is not considered a normal flora. However, it is not regarded as a pathogenic flora either, because the isolation rates of anaerobes (strongly associated with bacterial vaginosis) and fungi (occasionally causing vulvovaginal candidiasis) were lower than in the GBS-negative flora. These results suggest that the GBS-positive flora is associated with a lower risk of abnormality during pregnancy and abnormal pregnancy outcome compared with the GBS-negative flora, although this group is one of the most important pathogens in neonatal infections.