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The blue fox (<i>Vulpes lagopus</i>), a fur-bearing animal, is an important component of the breeding industry in China. Semen quality is a key factor for the reproductive process and the breeding effectiveness of the farmed blue fox. However, bacterial contamination in semen samples utilized in artificial fertilization is very common. The β-defensins, a class of important antimicrobial peptides in mammals, could protect the reproductive system of male animals from bacterial invasion, maintain the stability of the genital tract microenvironment and improve semen quality. In this study, molecular cloning and bioinformatics analysis were conducted to analyze the protein structure and function of blue fox β-defensin 108 (<i>Vulpes lagopus</i> beta-defensin 108, vBD108) and 122 (<i>Vulpes lagopus</i> beta-defensin 122, vBD122). To evaluate the bacteriostatic effect of recombinant vBDs (<i>Vulpes lagopus</i> beta-defensins) protein, varying concentrations (0, 25, 50, 100, 200 µg/mL) were taken to evaluate the effects on <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> at different times (0, 2, 4, 6, 8, 12 h). The results showed that vBD108 and vBD122 existed in different forms in protein structure and had antibacterial activity. Both proteins, at 50 µg/mL, had efficacious bacteriostatic activity. This study shows that recombinant vBD108 and vBD122 proteins have good antibacterial activity in vitro. This implies a potential role in improving semen quality and hygienic measures in the process of artificial insemination as an extender of semen dilution with antibacterial activity.