Vulvovaginal candidiasis (VVC) is a common vaginitis caused by Candida species,a frequently recurring condition. Fungal azole-resistant strains with azole-resistance have developed for long and wide explosion to the first-line antifungal azole agent. Bovine lactoferrin (BLF) is a protein from transferrin family secreted by the bovine mammary tissue. Its various biological functions are well known, especially the pronounced antifungal activity. In the current study, we constructed a Lactobacillus casei strain (L.casei/pPG612.1-BLF), which secreted BLF encoded by a mature secretion vector plasmid pPG612.1, and evaluated its antifungal activity in vitro and in vivo. In a two-layer agar plate in vitro assay, the number of C. albicans CFUs decreased and the average colony size shrunk upon exposure to L. casei/pPG612.1-BLF. In a murine VVC model, the infection burden of mice intra-vaginally pre-inoculated with L. casei/pPG612.1-BLF was lower than in control groups. Furthermore, the infection burden in mice with VVC was reduced when the animals were continually given L. casei/pPG612.1-BLF as a topical treatment for 5 days. Combined, these results suggested that the L. casei/pPG612.1-BLF strain is a promising preventative and therapeutic anti-VVC agent, highlighting the possibility of employing the probiotic L. casei as a vehicle for biotherapy in the female genital tract and exploiting the natural antibiotic antimicrobial peptides for other applications.