Malignant ascites frequently occur in patients with advanced ovarian cancer at initial diagnosis, and in almost all cases of relapse, they are closely related to poor prognosis, chemoresistance, and metastasis. To date, effective management strategies have been limited. In this study, we aimed to investigate the effects of oncolytic adenovirus (OV) on malignant ascites in a mouse model of advanced ovarian cancer. The results suggested that OV conferred an effective ability to reduce ascites development and prolong overall survival. Further analysis of the ascitic immune microenvironment revealed that OV treatment promoted T cell infiltration, activation, and differentiation into the effector phenotype; reprogrammed macrophages toward the M1-like phenotype; and increased the ratios of both CD8+ T cells to CD4+ T cells and M1 to M2 macrophages. However, immunosuppressive factors such as PD-1, LAG-3, and Tregs emerged after treatment. Combination therapy including OV, CSF-1R inhibitor PLX3397, and anti-PD-1 remarkably delayed the progression of ascites, and combination therapy induced a greater extent of T cell infiltration, proliferation, and activation. This study provides experimental and theoretical evidence for oncolytic virus-based treatment of malignant ascites, which may further contribute to advanced ovarian cancer therapy. Display omitted Effective management of ascites in ovarian cancer has been lacking. Here, we found that oncolytic adenovirus could treat ascites of ovarian cancer by reprogramming the ascitic immune microenvironment. Our study provides experimental and theoretical evidence for oncolytic virus-based treatment of malignant ascites, which may further contribute to advanced ovarian cancer therapy.