AbstractCertain anaerobic microbes with the capability to colonize the tumor microenvironment tend to express the heterologous gene in a sustainable manner, which will inevitably compromise the therapeutic efficacy and induce off‐tumor toxicity in vivo. To improve the therapeutic precision and controllability of bacteria‐based therapeutics, Escherichia coli Nissle 1917 (EcN), engineered to sense blue light and release the encoded flagellin B (flaB), is conjugated with lanthanide upconversion nanoparticles (UCNPs) for near‐infrared (NIR) nano‐optogenetic cancer immunotherapy. Upon 808 nm photoirradiation, UCNPs emit at the blue region to photoactivate the EcN for secretion of flaB, which subsequently binds to Toll‐like receptor 5 expressed on the membrane of macrophages for activating immune response via MyD88‐dependent signal pathway. Such synergism leads to significant tumor regression in different tumor models and metastatic tumors with negligible side effects. These studies based on the NIR nano‐optogenetic platform highlight the rational of leveraging the optogenetic tools combined with natural propensity of certain bacteria for cancer immunotherapy.