Cytoskeleton-associated protein 2-like (CKAP2L), a cell cycle-related protein, is correlated to tumor progression in some tumors. But there were no pan-cancer studies on CKAP2L, and its role in cancer immunotherapy is also unclear. The expression levels, expression activity, genomic alterations, DNA methylation and functions of CKAP2L in various tumors, as well as the associations between CKAP2L expression and patient prognosis, chemotherapy sensitivity, and tumor immune microenvironment, were all analyzed in a comprehensive pan-cancer analysis of CKAP2L by various databases, analysis websites, and R software. The experiments were also conducted to verify the analysis results. In the majority of cancers, CKAP2L expression and activity were markedly elevated. Elevated CKAP2L expression led to poor prognostic outcomes in patients, and is an independent risk factor for most tumors. Elevated CKAP2L causes decreased sensitivity to chemotherapeutic agents. Knockdown of CKAP2L significantly inhibited the proliferation and metastasis capacity of the KIRC cell lines and resulted in cell cycle G2/M arrest. In addition, CKAP2L was closely related to immune subtypes, immune cell infiltration, immunomodulators and immunotherapy markers (TMB, MSI), patients with high CKAP2L expression were more sensitive to immunotherapy in the IMvigor210 cohort. The results indicate that CKAP2L is a pro-cancer gene that serves as a potential biomarker for predicting patient outcomes. By inducing cells to transition from the G2 phase to the M phase, CKAP2L may promote cell proliferation and metastasis. Furthermore, CKAP2L is closely related to the tumor immune microenvironment and can be used as a biomarker to predict tumor immunotherapy.