Lactobacilli are predominant microorganism in vaginal ecology and provide protection against pathogens. The present study was aimed to evaluate the in vitro and in vivo prebiotic effect of pectin on vaginal lactobacilli. The effect of pectin concentration on growth of Lactobacilli casei (LC) and Lactobacilli fermentum (LF) was studied by measuring change in pH, optical density, titratable acidity, and dry mass after 48 h of incubation. The antimicrobial effect against pathogenic Escherichia coli and Candida albicans was studied by agar diffusion technique. In vitro effect of pectin on viability of mixed cultures of LC or LF with E. coli or C. albicans was studied. In vivo, prophylactic and therapeutic effect of pectin on vaginal lactobacilli was investigated on female Wistar rats. The stimulative effect of pectin was confirmed by an increase in LC and LF dry mass, reduction in pH, and increase in production of organic acid. Lactobacilli supernatants showed a significant antimicrobial effect against E. coli. The mixed culture study demonstrated the stimulative effect of pectin on LC and LF, whereas inhibitory effects on E. coli and C. albicans. In vivo study on female rats revealed a significant increase in lactobacilli count in vaginal flora after topical administration of pectin. This study demonstrated the prophylactic and therapeutic effect of pectin as a prebiotic on vaginal microflora.