Lactobacillus plays an important role in keeping the vaginal micro-ecological balance and inhibiting the growth and reproduction of pathogens. However, the knowledge of Lactobacillus distributions and functions in pregnant women remains unclear. The study was undertaken the prevalence of Lactobacillus and the specific strains of Lactobacillus present in vaginal discharge from women at different stages of pregnancy. This study was performed between October 2009 and January 2010 at the Department of Obstetrics and Gynecology, Beijing Friendship Hospital, Affiliate of Capital University of Medical Sciences, China. pH value of vaginal discharges was determined, vaginal discharges were collected and Lactobacillus were isolated and cultured, hydrogen peroxide generation of Lactobacillus was performed on 120 pregnant women. Statistical analysis was carried out using SPSS 11.5 software. Among the 120 samples of vaginal discharge, Lactobacilli was isolated in 104 samples (86.7%), 80 were Lactobacillus acidophilus (76.9%), 80 were LB+ (76.9%), 64 were L. acidophilus. Most L. acidophilus isolates could produce hydrogen peroxide. pH gradually decreased with ongoing pregnancy, but there were no significant differences among the three trimesters (P>0.05). Our results indicated that L. acidophilus is the dominant bacterial strain in terms of the vaginal Lactobacillus distributions and functions in pregnant women.