BACKGROUND: 1,25(OH)(2)D(3) supplementation alone does not provide sufficient benefit to hepatocellular carcinoma (HCC) patients in clinical trials. Tumor-associated macrophages (TAMs)-mediated immunosuppression is regarded as a major hurdle for the effectiveness of several treatments. Previous studies revealed that hCAP18/LL-37 was an important factor which directly suppresses the anticancer activity of 1,25(OH)(2)D(3) on HCC cells. However, whether TAMs contribute to the limited clinical efficacy of 1,25(OH)(2)D(3) through hCAP18/LL-37 remains unclear. METHODS: Co-culture systems of HCC cells (PLC/PRF-5, Huh7) with THP-1-derived macrophages and co-xenograft mouse models were established. Anticancer activity was evaluated in vitro and in vivo mouse models using standard assays. Mechanistic investigations utilized qRT-PCR, Western blot, flow cytometry, ELISA, and immunohistochemistry. Therapeutic efficacy of 1,25(OH)(2)D(3)/suramin combination was assessed in co-xenograft and N-Nitrosodiethylamine (DEN)/Carbon tetrachloride (CCl(4))-induced HCC models. RESULTS: 1,25(OH)(2)D(3) (200-500 nM) promoted macrophage recruitment, M2 polarization, Akt/mTOR signal and STAT3 signal activation in HCC/macrophage co-culture systems. This effect was mediated by 1,25(OH)(2)D(3)-induced hCAP18/LL-37 overexpression, which facilitated TAM infiltration and M2 reprogramming. Suramin, a potent LL-37 inhibitor, abrogated these immunosuppressive effects by blocking LL-37 internalization, restoring M1 polarization and suppressing Akt/mTOR and STAT3 pathways. Notably, 1,25(OH)(2)D(3)/suramin combination therapy synergistically inhibited HCC proliferation, colony formation, and invasion in vitro. In xenograft models and DEN/CCl(4)-induced HCC models, suramin enhanced 1,25(OH)(2)D(3)'s efficacy by promoting M1 polarization, increasing intratumoral M1/M2 ratios, reducing tumor growth, and diminishing macroscopic nodules. CONCLUSION: The 1,25(OH)(2)D(3)-LL-37-TAM axis drives immunosuppression in HCC by modulating macrophage phenotypes. While suramin potently disrupts this axis, blocking LL-37-mediated TAMs recruitment and M2 polarization, while promoting antitumor M1 phenotype responses. These findings highlight suramin as a promising adjunct to 1,25(OH)(2)D(3)-based immunotherapy for HCC.