BACKGROUND: Immune checkpoint inhibitors are effective to treat hepatocellular carcinoma (HCC) yet only successful in a small part of patients. This study aimed to investigate whether poly-ICLC, an immune stimulant, can enhance the antitumor effects of anti-PD-1 on mouse HCC. METHODS: We established two syngeneic HCC mouse models with BNL cells in BALB/c mice and Hep-55.1 C cells in C57BL/6 J mice. Mice with subcutaneous HCC tumors received one of five treatments: control, anti-PD-1, intratumoral (IT) poly-ICLC, anti-PD-1 plus intramuscular (IM) poly-ICLC, or anti-PD-1 plus IT poly-ICLC. Tumor volumes were measured, CD8+ T lymphocytes in tumors and spleen were analyzed, and interferon-γ activity was assessed by ELISpot. Immune cell types and abundance were evaluated with NanoString nCounter IO360 panels. RESULTS: Cotreatment with poly-ICLC significantly enhanced the antitumor effects of anti-PD-1, with IT administration being more effective than IM. IT poly-ICLC also induced more significant CD8 CONCLUSIONS: Combination therapy with poly-ICLC, especially through IT route, and anti-PD-1 provides significantly greater antitumor effects than anti-PD-1 monotherapy in syngeneic mouse models of HCC.