Hepatocellular carcinoma (HCC) is a malignant tumor, making up about 90% of all primary liver cancers and ranking as a top cause of cancer mortality globally. With the severe side effects of conventional radiotherapy and chemotherapy, there's an ongoing quest for less toxic treatment options. CD24, a highly glycosylated membrane surface protein, plays a role in cancer cell adhesion and metastasis. Its overexpression is associated with poor prognosis in cancer patients. By binding to Siglec-10 on macrophages, CD24 helps tumor cells dodge the immune system. Targeting CD24 expression on is seen as a promising approach to cancer therapy. This study investigated the potential of repurposing the old drug erythromycin to regulate CD24 and its anti-HCC effect. The results investigated that erythromycin didn't significantly impact liver cancer cell growth, movement, or death. However, it effectively inhibited CD24 expression after treatment, and the effect that can be reversed with a proteasome inhibitor MG132. Further study showed erythromycin not only reduced CD24 expression but also slowed tumor growth, triggered cell death, and altered the balance of proteins involved in cell proliferation and migration in vivo. Importantly, it altered the immune environment within tumors and boosted the presence of immune cells that fight cancer including the infiltration increase of M1 macrophages and T lymphocytes in tumor tissues and the decrease of M2 macrophages. The research confirmed that erythromycin enhanced the anti-tumor immune response in HCC-bearing mice by reducing CD24 expression, providing a novel strategy for the clinical development of HCC treatments.