Objective: Melanoma is one of the human malignant tumors with high drug resistance and difficult to treat. Ferroptosis is a new type of programmed cell death, and inducing ferroptosis is a new strategy for the treatment of melanoma. Annexin A5 (ANXA5) has been shown to be abnormally expressed in many types of tumors. This project is to explore the correlation between ANXA5 and ferroptosis in melanoma and provide guidance for melanoma treatment targeting ferroptosis. Methods: Based on the Human Protein Atlas database, ANXA5 expression in tissues and tumors were systematically analyzed. Human melanoma sections were used for detecting ANXA5 by immunohistochemical analysis. Knockout of AnxA5 was constructed in melanoma cell line B16F10 by a CRISPR/Cas9 system, then the effect of ANXA5 on ferroptosis was determined by CCK8 and LDH assay in B16F10 cells. RNA-seq was further performed to identify the possible target molecules of ANXA5 related to ferroptosis. Finally, the regulatory role of ANXA5 on ferroptosis sensitivity was detected in different melanoma cells. Results: ANXA5 is abnormally high expressed in melanoma, and knockdown or knockout of ANXA5 promoted the sensitivity to ferroptosis in melanoma cells. Acyl-CoA synthetase long-chain family member 4 (ACSL4) as a key regulator of ferroptosis,was negatively correlated with ANXA5 expression. Overexpression of ANXA5 reduced the level of ACSL4 and showed resistance to ferroptosis. Conclusion: In melanoma cells, knockdown of ANXA5 enhanced the sensitivity to ferroptosis via upregulating ACSL4, suggesting that ANXA5 might be a potential target of ferroptosis for melanoma therapy.