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Drugs selectively targeting replication stress have demonstrated significant preclinical activity, but this has not yet translated into an effective clinical treatment. Here we report that targeting increased replication stress with a combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea targets also promotes pro-inflammatory cytokine/chemokine expression that is independent of cGAS-STING pathway activation and immunogenic cell death in human and murine melanoma cells. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. It increases cytotoxic CD8+ T cell activity, but the major adaptive immune response is a pronounced NKT cell tumour infiltration. Treatment also promotes an immunosuppressive tumour microenvironment through CD4+ Treg and FoxP3+ NKT cells. The number of these accumulated during treatment, the increase in FoxP3+ NKT cells numbers correlates with the decrease in activated NKT cells, suggesting they are a consequence of the conversion of effector to suppressive NKT cells. Whereas tumour infiltrating CD8+ T cell PD-1 and tumour PD-L1 expression was increased with treatment, peripheral CD4+ and CD8+ T cells retained strong anti-tumour activity. Despite increased CD8+ T cell PD-1, combination with anti-PD-1 did not improve response, indicating that immunosuppression from Tregs and FoxP3+ NKT cells are major contributors to the immunosuppressive tumour microenvironment. This demonstrates that therapies targeting replication stress can be well tolerated, not adversely affect immune responses, and trigger an effective anti-tumour immune response.