Introduction: Mucosal macrophages are capable of exhibiting immune activatory or suppressive responses to pathogenic or commensal bacteria, respectively. Such responses are dependent on stimulus received and macrophage subset, where tolerance is associated with a homeostatic M2-like subset and immune activation/inflammatory responses are associated with M1-like macrophages. Lactobacillus plantarum and Lactobacillus salivarius are probiotic bacteria, which exhibit immunomodulatory capabilities in determining activation/suppression to microbial challenge. Research suggests that dysbiosis and dysregulation of mucosal tolerance underlies the pathological mechanisms driving autoimmune disease. Understanding the role of such mechanisms and how they can be manipulated by probiotics may have a profound effect on controlling mucosal pathology. The aim of this study was to investigate the immunomodulatory role of selected probiotic strains on controlling immune activation versus suppression in distinct macrophage subsets. Methods: M1 and M2-like macrophages were generated from the THP-1 monocytic cell line by differentiation with PMA or Vitamin D3 for 4 and 8 days, respectively. Macrophages were then subjected to defined pre-stimulation/stimulation protocols using E.coli K12 LPS or the probiotic strains Lactobacillus plantarum C28 and Lactobacillus salivarius MS13, afterwhich cytokine secretion was analysed by sandwich ELISA. Results: LPS endotoxin tolerisation suppressed both M1 and M2-TNFa secretion, whereas C28 and MS13 only suppressed M2-TNFa. With respect to inflammasome-dependent cytokines, LPS failed to suppress IL-1b and IL-18. C28 however, failed to suppress either IL-1b or IL-18 in both macrophage subsets yet primed/augmented IL-1b in M1 and M2s. On the other hand, MS13 augmented M1-IL-18 and suppressed M2-IL-1b. Conclusion: The probiotic strains, MS13 and C28 differentially primed and suppressed macrophage cytokine secretion in a subset-specific manner, where MS13 favoured a tolerogenic/homeostatic function and C28 selectively activated inflammatory responses.