We investigated the role of TONSL, a mediator of homologous recombination repair (HRR), in stalled replication fork double-strand breaks (DSBs) in cancer. Publicly available clinical data (tumors from the ovary, breast, stomach and lung) were analyzed through KM Plotter, cBioPortal and Qomics. Cancer stem cell (CSC)-enriched cultures and bulk/general mixed cell cultures (BCCs) with RNAi were employed to determine the effect ofTONSLloss in cancer cell lines from the ovary, breast, stomach, lung, colon and brain. Limited dilution assays and ALDH assays were used to quantify the loss of CSCs. Western blotting and cell-based homologous recombination assays were used to identify DNA damage derived from TONSL loss.TONSLwas expressed at higher levels in cancer tissues than in normal tissues, and higher expression was an unfavorable prognostic marker for lung, stomach, breast and ovarian cancers. Higher expression ofTONSLis partly associated with the coamplification ofTONSLandMYC, suggesting its oncogenic role. The suppression ofTONSLusing RNAi revealed that it is required in the survival of CSCs in cancer cells, while BCCs could frequently survive withoutTONSL.TONSLdependency occurs through accumulated DNA damage-induced senescence and apoptosis inTONSL-suppressed CSCs. The expression of several other major mediators of HRR was also associated with worse prognosis, whereas the expression of error-prone nonhomologous end joining molecules was associated with better survival in lung adenocarcinoma. Collectively, these results suggest that TONSL-mediated HRR at the replication fork is critical for CSC survival; targeting TONSL may lead to the effective eradication of CSCs.