Purpose <p>Never in mitosis gene A-related kinase 6 (NEK6) is involved in mitotic cell cycle. The objective of the present study is to comprehensively explore the prognostic value of NEK6 and its potential functions in multiple cancers, especially in gliomas.</p> Methods <p>We performed comprehensive analyses of NEK6 in pan-cancer, including expression profile, immune characteristics and its relationship with clinical prognosis. Moreover, we validated our significant conclusions through multiple cell experiments (such as clone formation assay, MTT assay and flow cytometry) and analyses based on clinical samples (such as immunohistochemistry analysis).</p> Results <p>NEK6 was significantly upregulated in gliomas. And our study indicated that the increased level of NEK6 was associated with poor clinical prognoses of patients. The single-cell analysis revealed that NEK6 overexpression was highly related to malignant cells and Mono/Macrophages (denoted as the monocyte and macrophage population) in glioma tissue. The decrease in NEK6 hindered the growth and migration capacity of the glioma cells, leading to a halt in the G2/M phase of the cell cycle and triggering apoptosis.</p> Conclusion <p>Taken together, our data uncovered the prognostic value, therapeutic potential, and molecular insight of NEK6 in glioma.</p>