Glioblastoma (GBM) is the most common and deadly malignant primary brain tumor. Chromobox (CBX) family proteins are essential components of the epigenetic regulatory complex and are involved in the occurrence and development of various cancers. However, the roles of CBX members in GBM is little known. In this analysis, we synthesized several mainstream bioinformatics databases to comprehensively explore the expression profiles, prognostic implications, genetic alterations, immune infiltration, and potential biological functions of the CBXs in GBM, and cell experiments were also conducted to investigate the role of CBX8 in GBM. We found that the elevated mRNA expression of CBX2/3/5/8 and reduced mRNA expression of CBX6/7 were found in GBM. The protein levels of CBX2/3/5/8 were elevated in GBM tissues, whereas the protein levels of CBX6/7 showed no significant difference. The upregulated expression of CBX2/3/8 was found to be both correlated with the tumor grade and recurrent status. The overexpression of CBX3/8 and underexpression of CBX6 mRNA were associated with the poor prognosis. These findings suggested that CBX3 and CBX8 might be useful diagnostic and prognostic biomarkers in GBM. Further cell experiment results supported that CBX8 promoted the proliferation of glioma cells. Moreover, a high genetic alteration rate of CBXs (37%) was found in GBM and to varying degrees. The expression of CBXs was significantly related to the immune cells infiltration. CBX7 methylation level was significantly increased in GBM tissues. Our results may provide novel ideas to find potential prognostic markers and new therapeutic targets among CBX family members in glioblastoma.