Background <p>Cancer stem cells (CSCs) are considered the ‘seeds’ of recurrence after chemotherapy, but eliminating CSCs remains notoriously challenging. This study aims to examine whether cell cycle checkpoint kinase 1 (CHK1) blockade can abrogate the stemness of ovarian cancer (OC) cells, making them easier targets of anti-tumor immunity.</p> Methods <p>Prexasertib was used to block CHK1 in OC cell lines and xenografts, and its cytotoxicity was assessed <i>in vitro</i> and <i>in vivo</i>. <i>In vitro</i> tumor-sphere formation assays and stemness markers were used to evaluate cell stemness. PD-L1 expressions were examined via qRT-PCR, Western blot, flow cytometry, and immunohistochemistry. Prexasertib in combination with anti-PD-L1 antibody Atezolizumab was tested in immune-proficient mice bearing OC xenografts in terms of effects on tumor growth, tumor cell stemness, and tumor infiltrating lymphocytes via tumor volume monitoring, immunohistochemistry, and flow cytometry.</p> Results <p>Prexasertib effectively inhibited CHK1 phosphorylation, exhibited significant anti-tumor effects <i>in vitro</i> and <i>in vivo</i>, accompanied by decreased OC cell stemness. CHK1 was highly expressed in tumor spheres versus tumor cells cultured in 2D system, and Prexasertib treatment suppressed sphere formation and reduced the ALDH⁺ cell fraction. Unexpectedly, Prexasertib upregulated PD-L1 expression in tumor cells. <i>In vivo</i>, combining Prexasertib with Atezolizumab led to more remarkable remission of tumors, when compared with Prexasertib or Atezolizumab alone. Meanwhile, the tumor-infiltrating CD8⁺ T cells significantly increased in the combination group, while exhausted T cells decreased; the treatments did not affect CD4⁺ cell infiltration.</p> Conclusion <p>Dual targeting of CHK1 and PD-L1 may improve OC treatment by simultaneously suppressing stemness and enhancing anti-tumor immunity.</p>