BACKGROUND An increasing number of studies have demonstrated that CX3CL1 is involved in the development of tumors and may thus be considered a new potential therapeutic target for them. However, the function of CX3CL1 in clear cell renal cell carcinoma (ccRCC) remains poorly defined. METHODS The pan-cancer expression pattern and prognostic value of CX3CL1 were evaluated in this study. Moreover, the relationship of CX3CL1 expression with the tumor microenvironment, especially the tumor immune microenvironment, was analyzed. Our analyses employed public repository data. Additionally, we generated stable CX3CL1-overexpressing 786-O cells to determine the role of CX3CL1 in vitro via cell viability and transwell assays. A xenograft tumor model was used to determine the role of CX3CL1 in vivo. The association between CX3CL1 and ferroptosis sensitivity of tumor cells was assessed using Ferrostatin-1. RESULTS Our findings indicated the involvement of CX3CL1 in the occurrence and development of ccRCC by acting as a tumor suppressor. We also found that ccRCC patients with high CX3CL1 expression showed better clinical outcomes than those with low CX3CL1 expression. The findings of our epigenetic study suggested that the expression of CX3CL1 in ccRCC is correlated with its DNA methylation level. Furthermore, the CX3CL1 expression level was closely related to the infiltration level of CD8+ T cells into the tumor microenvironment (TME). CX3CL1 showed different predictive values in different immunotherapy cohorts. Finally, CX3CL1 overexpression inhibited tumor cell proliferation and metastasis and promoted tumor ferroptosis sensitivity in ccRCC. CONCLUSIONS This study revealed the role of CX3CL1 as a tumor suppressor in ccRCC. Our findings indicated that CX3CL1 plays a crucial role in regulating the ccRCC TME and is a potential predictor of immunotherapy outcomes in ccRCC. We also found that CX3CL1 can promote ferroptosis sensitivity in ccRCC cells.