Background and Aims As is widely known, the microbiota in the gastrointestinal tract plays an important role in regulating intestinal health. Having a balanced microbiota can help prevent diseases, particularly cancer related to the gastrointestinal system. Lactobacillus rhamnosus (L. rhamnosus) has been found to have anti-tumor effects, but the specific mechanisms are still unclear. Based on this, this study was conducted to investigate the impact and potential mechanisms of L. rhamnosus on apoptosis of human colorectal cancer cells (CRC).Methods Human CRC cells HCT-116, HT-29, and normal colon epithelial cells NCM-460 were treated with the culture supernatant of L. rhamnosus (LRCS) or Escherichia coli (E. coli) culture supernatant (ECCS), and cell viability, apoptosis level, and cell cycle distribution were measured. The bacterial culture supernatant was divided into low molecular weight (<3 kDa) and high molecular weight (>3 kDa) fractions using a 3 kDa ultrafiltration membrane, and the effects of these fractions on cell apoptosis were analyzed. Untargeted LC-MS/MS was used to identify anti-CRC metabolites in the effective fraction of LRCS, and these metabolites were screened at a concentration of 5 μmol/L to identify key metabolites affecting apoptosis. SiRNA screening was performed to identify the target of the key metabolites in the apoptotic pathway. Molecular docking analysis was conducted to study the interaction sites between the key metabolites and the target molecules.Results LRCS exhibited a concentration-dependent decreasing effect on cell viability of CRC cells and significantly promoted apoptosis in CRC cells (all P<0.05), while ECCS had no such effects (all P>0.05). Further analysis revealed that only the low molecular weight fraction of LRCS was capable of producing these effects. Untargeted LC-MS/MS identification and validation experiments indicated that indole-3-lactic acid (I3L) was a key metabolite of L. rhamnosus that influenced CRC apoptosis. Through siRNA screening, I3L showed no significant effect on apoptosis level or TNF-α expression and secretion levels in CRC cells with knockout of the specific protein 3 (SP3) (all P>0.05). Molecular docking revealed that I3L interacted with K551, E551, and E552 of SP3. CRC cells with SP3 knockout were transfected with wild-type SP3 overexpression plasmid and mutant SP3 overexpression plasmid, respectively, and I3L was found to promote cell apoptosis and TNF-α secretion in the former (all P<0.05), but had no effect on those in the latter (all P>0.05).Conclusions L. rhamnosus can promote apoptosis of CRC cells, and the mechanism of action may be related to its metabolite I3L binding to the K551, E551, and E552 sites of SP3, leading to increased transcription and secretion of TNF-α.