Preventing tumor recurrence after radiofrequency ablation (RFA) of malignant solid tumors with large size or in high-risk locations represents a great challenge. In this study, we explored the feasibility of using oncolytic peptide LTX-315 plus an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody for inhibiting residual tumors after RFA of hepatocellular carcinoma (HCC). In in vitro experiment, the CD8T cells from Hepa1-6 tumors, after being subjected to three different treatments (control, iRFA, iRFA + LTX-315), were extracted and were then co-cultured with Hepa1-6 cells and an anti-CTLA-4 antibody. The enzyme-linked immunospot, flow cytometry, and cell counting kit-8 assay were employed to assess the cytotoxicity of extracted CD8T cells on Hepa1-6 cells. In in vivo experiment, different murine orthotopic HCC models were variously treated by: (1) pseudo iRFA + phosphate-buffered saline (PBS); (2) iRFA + PBS; (3) iRFA + LTX-315; (4) iRFA + anti-CTLA-4 antibody; and (5) iRFA + LTX-315 + anti-CTLA-4 antibody. The treatment effects were compared among different groups and were pathologically confirmed. The possible mechanisms of the combination treatment (LTX-315+anti-CTLA-4 antibody) for residual tumors after iRFA of HCC were explored. LTX-315 significantly reduced the PD-1 expression and significantly increased CTLA-4 expression of CD8T cells in residual tumors, and additional treatment of anti-CTLA-4 antibody could significantly enhance the cytotoxicity of CD8T cells for Hepa1-6 cells in vitro experiments. Compared with the other treatments, the combined treatment of LTX-315 with anti-CTLA-4 antibody achieved a better tumor response and longer survival, and it could synergistically activate the cGAS-STING pathway and elicit an immunogenic cell death, leading to a strong anti-tumor immunity after iRFA of HCC. The immunosuppressive microenvironment of residual tumors was significantly improved by the combination therapy with a significantly increased ratio of M1-like tumor-associated macrophages to M2-like tumor-associated macrophages, a significantly decreased infiltration of regulatory T cells and myeloid-derived suppressor cells, and a significantly lower expression of PD-1 and CTLA-4. Overall, the results of this study demonstrated that LTX-315 plus anti-CTLA-4 antibody could synergistically improve the immunosuppressive microenvironment of residual tumors and induce a strong anti-tumor immunity after iRFA of HCC. This combination treatment strategy may offer a new alternative to reduce the tumor recurrence after RFA of malignant solid tumors with large sizes or in high-risk locations.