Aims We investigated the ability of Lactococcus lactis, a species generally regarded as safe, to express tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. The expressed protein was either cell wall anchored or secreted, and it was assessed whether this could induce apoptosis in human colon adenocarcinoma cell lines SW480 and HCT116. Methods and Results Constructs were designed to produce either secreted or cell wall-anchored forms of human TRAIL cloned into pNZ7021 expression vector. The expression by L. lactis was confirmed by Western blotting and immunofluorescence. Induction of cell death was evaluated by coculturing transformants producing either form of TRAIL protein with the two cell lines followed by MTT assay. Gene expression of apoptosis genes, Bax and Bcl2, was assessed by qPCR. The viability of SW480 and HCT116 cells treated with recombinant L. lactis was significantly reduced. A significant change was observed in the ratio of Bax/Bcl2 expression in HCT116 cells only following treatment with the supernatant of recombinant L. lactis containing secreted TRAIL. Conclusion Recombinant L. lactis producing TRAIL protein can induce apoptosis in human colon adenocarcinoma cell lines SW480 and HCT116. Significance and Impact of the Study The use of recombinant probiotics that produce anticancer compounds is a promising option for combating cancer cells.