The molecular mechanisms of pro-apoptotic effects of human-derived Lactobacillus reuteri ATCC PTA 6475 were investigated in this study. L. reuteri secretes factors that potentiate apoptosis in myeloid leukemia-derived cells induced by tumour necrosis factor (TNF), as indicated by intracellular esterase activity, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assays and poly (ADP-ribose) polymerase cleavage. L. reuteri downregulated nuclear factor-κB (NF-κB)-dependent gene products that mediate cell proliferation (Cox-2, cyclin D1) and cell survival (Bcl-2, Bcl-xL). L. reuteri suppressed TNF-induced NF-κB activation, including NF-κB-dependent reporter gene expression in a dose-and time-dependent manner. L. reuteri stabilized degradation of IκBα and inhibited nuclear translocation of p65 (RelA). Although phosphorylation of IκBα was not affected, subsequent polyubiquitination necessary for regulated IκBα degradation was abrogated by L. reuteri. In addition, L. reuteri promoted apoptosis by enhancing mitogen-activated protein kinase (MAPK) activities including c-Jun N-terminal kinase and p38 MAPK. In contrast, L. reuteri suppressed extracellular signal-regulated kinases 1/2 in TNF-activated myeloid cells. L. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IκBα ubiquitination and enhancing pro-apoptotic MAPK signalling. An improved understanding of L. reuteri-mediated effects on apoptotic signalling pathways may facilitate development of future probiotics-based regimens for prevention of colorectal cancer and inflammatory bowel disease.