Background: Anlotinib, an oral multi-targeted receptor tyrosine kinase inhibitor (TKI), exhibits good safety and efficacy in many advanced refractory solid tumors. Previous studies have indicated that anlotinib has synergistic effects with anti–programmed death-1 antibodies via modulation of the tumor microenvironment. Our study aims to examine the efficacy and safety of anlotinib-pembrolizumab combination therapy as a treatment for refractory or recurrent High-Grade Serous Ovarian Cancer. Methods: Patients with refractory or recurrent High-Grade Serous Ovarian Cancer received pembrolizumab (200 mg, intravenously over 60 minutes, once every 3 weeks) plus anlotinib (12 mg/day, orally, 2 weeks on and 1 week off, every 3 weeks) therapy or pembrolizumab (200 mg, intravenously over 60 minutes, once every 3 weeks) monotherapy, respectively. Primary end points were progression-free survival (PFS) and overall survival (OS). The relationships of potential biomarkers with clinical efficacy were also evaluated. Results: A total of 33 patients were enrolled, with 15 participants received the anlotinib-pembrolizumab combination regime and 18 with pembrolizumab monotherapy, respectively. Grade 3 treatment-related adverse events (TRAEs) were recorded in 3 participants (20%) receive the combination treatment and 3 (16.7%) in the pembrolizumab monotherpay group, respectively. The PFS were 13.0 months and 8.0 months to anlotinib-pembrolizumab combination regime and pembrolizumab mono-treatment, respectively. And the OS of the two groups were equal as 31 months. In the anlotinib-pembrolizumab combination treatment group, patients with ARID1A mutation indicated a significantly survival benefit compared patient with the wide type (PFS: 12.5 VS. 7.0, P = 0.004 ). Conclusions: The anlotinib-pembrolizumab combination showed promising efficacy and favorable safety as treatment for refractory or recurrent High-Grade Serous Ovarian Cancer. The ARID1A are potential biomarkers for predicting the efficacy of this novel regimen.