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Abstract Understanding the immunological effects of chemotherapy is of great importance, especially now that we have entered an era where ever-increasing pre-clinical and clinical efforts are put into combining chemotherapy and immunotherapy to combat cancer. Single-cell RNA sequencing (scRNA-seq) has proved to be a powerful technique with a broad range of applications, studies evaluating drug effects in co-cultures of tumor and immune cells are however scarce. We treated a co-culture comprised of human colorectal cancer (CRC) cells and peripheral blood mononuclear cells (PBMCs) with the nucleoside analogue trifluridine (FTD) and used scRNA-seq to analyze posttreatment gene expression profiles in thousands of individual cancer and immune cells concurrently. ScRNA-seq recapitulated major mechanisms of action previously described for FTD and provided new insight into possible treatment-induced effects on T-cell mediated antitumor responses.