Mesenchymal stem cells (MSCs) hold therapeutic promise for treating inflammatory bowel disease (IBD) owing to their immunomodulatory properties. Currently, pre-conditioning strategies with several beneficial agents have been applied to enhance the efficacy of MSCs in treating IBDs. Probiotics are increasingly acknowledged as supplemental therapy for IBD; however, their potential benefits in MSCs-based therapy remain largely unexplored. In this study, we hypothesized that pretreating MSCs with Lactobacillus sakei CVL-001 (L. sakei CVL-001), a representative probiotic strain, could improve their therapeutic effectiveness for IBD. In line with this hypothesis, we noted that pretreatment with L. sakei CVL-001 significantly induced IL-10 secretion in MSCs via the activation of the STAT3 signaling pathway. These primed MSCs reduced pro-inflammatory cytokine production in LPS/IFN-γ-treated macrophages and promoted an M2 phenotype, associated with immunoregulation and tissue repair, in undifferentiated macrophages. In addition, their conditioned media significantly reduced the proliferation capacity of Jurkat T cells and splenocytes, while the neutralization of IL-10 reversed these phenomena. Furthermore, MSCs treated with L. sakei CVL-001 mitigated inflammatory responses and promoted epithelial regeneration, leading to accelerated recovery from disease symptoms and improved survival rates compared to naive MSCs in a DSS-induced colitis mouse model. In conclusion, our findings suggest that probiotics, such as L. sakei CVL-001, can improve the therapeutic efficacy of MSCs for treating IBD.