Purpose C motif chemokine ligand 21 (CCL21) is important for tissue homeostasis, immune surveillance. However, the relationship between CCL21 expression, tumor immunity, and prognosis in human tumors remains unclear. Methods CCL21 expression data were extracted from online databases, including The Cancer Genome Atlas (TCGA), Genotype Tissue Expression (GTEx) and Cancer Cell Line Encyclopedia (CCLE). The Tumor IMmune Estimation Resource (TIMER) database was used to study the association between CCL21 expression and tumor immune infiltration and immune checkpoint genes. Pancancer correlation analysis of CCL21 expression with the tumor-infiltrating immune response subtype was performed by TISIDB. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and the STRING tool were used to elucidate the potential mechanism of CCL21 in human tumorigenesis. Results CCL21 expression was significantly lower in most human tumors than in corresponding normal tissues and correlated with prognosis. CCL21 expression showed a strong positive correlation with tumor-infiltrating B cells, CD4+ and CD8+ T cells, macrophages, neutrophils, and dendritic cells. CCL21 also correlated positively with common immune checkpoint markers, including PD-1, TIM-3 and CTLA-4. Moreover, we found that CCL21 was an important regulatory factor participating in many signaling events, especially cytokine‒cytokine interactions and chemokine signaling pathways. Conclusions CCL21 is generally expressed at low levels among various types of cancers and may have an important influence on tumorigenesis, which is significantly associated with immune checkpoint biomarkers and tumor immunity. Together, these findings suggest that CCL21 is a prognostic biomarker that determines cancer progression and may serve as a therapeutic target. Keywords CCL21, prognosis, pancancer analysis, immunity, immunotherapy, therapeutic target