BACKGROUND: The therapeutic targeting of the intestinal microbiota has gained increasing attention as a promising avenue for addressing mood disorders. This study aimed to assess the potential effect of supplementing standard pharmacological treatment with the probiotic kefir in patients with Major Depressive Disorder (MDD). METHODS: Thirty-eight female participants diagnosed with moderate MDD by the Hamilton Rating Scale for Depression (HAM-D) were selected to receive the probiotic kefir in conjunction with antidepressant therapy for 12 weeks. The participants were evaluated at baseline (T0) and 90 days after probiotic kefir supplementation (T90). HAM-D scores and blood samples were collected at both time points. RESULTS: Probiotic supplementation significantly reduced MDD severity, as evidenced by lower HAM-D scores compared to baseline. Probiotic consumption for 90 days also significantly decreased interleukin-6 (IL-6), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) levels compared to baseline. However, probiotic kefir supplementation did not significantly affect serum serotonin levels. Additionally, after 90 days of probiotic consumption, insulin and morning cortisol levels were significantly reduced. In contrast, no significant changes were observed in serum levels of prolactin, vitamin D, and afternoon cortisol. CONCLUSION: This study provides valuable insights into the potential benefits of probiotics, specifically kefir, as adjunctive therapy for female patients with MDD. The findings highlight promising results in ameliorating depressive symptoms and modulating inflammatory and hormonal markers.