BACKGROUND: Currently, the use of probiotics to treat inflammatory bowel diseases (IBD) is widely accepted because of their gut microbiota modulation capabilities and anti-inflammatory potential. OBJECTIVE: The aim of this study is to examine the immunomodulatory outcomes of probiotics and sulfasalazine in the acetic acid-induced colitis murine model. METHODS: The animals were randomly assigned to one of the seven groups. Following the induction of colitis, Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12, and sulfasalazine (SASP) were orally administered for 10 days. Subsequently, the in vitro anti-inflammatory effect on TNF-alpha and IL-10 in the supernatants of cultured spleen cells was assessed via ELISAs. Relative mRNA expression of ZO-1, MLCK, iNOS, TNFR2, ROR-gammat, GATA-3, T-bet, and Foxp3 was determined using quantitative reverse‑transcription polymerase chain reaction (qRT‑PCR). RESULTS: The SASP plus probiotic mixture was more effective in alleviating colitis symptoms, and reducing disease activity scores, and mucosal inflammation. qRT-PCR analysis revealed a significant reduction in T-bet and RORgammat levels, while Foxp3 and GATA-3 levels increased in the colons of colitis mice. In addition, the selected strains substantially inhibited the release of inflammatory markers. Administration of LA-5 + BB-12 + SASP resulted in considerably higher inhibition of NO production and cell proliferation than in the other groups (p < 0.001). Treatment with LA-5 + BB-12 + SASP also reduced TNF-alpha-mediated apoptosis in intestinal epithelial cells (IECs). CONCLUSIONS: Survey results highlight that the combination regimen could be a promising strategy for IBD therapy, warranting further study of its clinical application and long-term benefits.