To investigate the expression levels of marginal zone B and B1-cell-specific protein (MZB1) in pan-cancer and its association with patient prognosis and tumor microenvironment (TME).MZB1 expression data, clinicopathological parameters, and survival data from 33 cancer types were extracted from the UCSC database for analyzing the correlations of MZB1 with clinical stage, patient prognosis, immunomodulatory genes, immune checkpoint genes, tumor stemness, immune cell infiltration, tumor mutational burden (TMB), and microsatellite instability (MSI). MZB1 gene mutations in pan-cancer were assessed using cBioPortal online database, and the value of MZB1 for cancer diagnosis was evaluated using ROC curve analysis. MZB1 expression levels in myeloid leukemia and renal carcinoma cells were detected using RT-qPCR and Western blotting, and the effect of MZB1 knockdown on cell proliferation was examined using EdU assay.MZB1 was significantly overexpressed in 20 cancer types, including kidney renal clear cell carcinoma (KIRC), breast invasive carcinoma, and acute myeloid leukemia. Its expression was associated with TNM stage, clinical stage, overall survival, and progression-free survival in multiple cancers. In most tumors, MZB1 expression was correlated significantly with immunomodulatory genes, immune checkpoint genes, tumor stemness, immune cell infiltration, TMB, and microsatellite instability. Gene amplification was the predominant mutation type of MZB1 in pan-cancer, and MZB1 showed high diagnostic value for skin cutaneous melanoma, KIRC, and head and neck squamous cell carcinoma. MZB1 was highly expressed in different myeloid leukemia cell lines and renal carcinoma cell lines, and MZB1 knockdown significantly suppressed the proliferation of HL60 and 769-P cells.MZB1 is highly expressed in a variety of tumors, and its aberrant expression affects the occurrence and prognosis of many tumors, suggesting its potential as a novel tumor biomarker and immunomodulatory target.