Gliomas are primary brain tumors known for their resistance to radiotherapy and frequent recurrence. This might result from the high heterogeneity and transcriptional plasticity of gliomas. Heat shock proteins are associated with unfavorable tumor outcomes and protect tumors from the effects of radiotherapy. However, their influence on brain tumors is not fully understood. Initial analyses of glioma patients from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases who had undergone radiotherapy identified HSP90B1 as a crucial gene affecting patient prognosis. Subsequent investigations revealed that HSP90B1 enhanced the proliferation, migration, and invasion of glioma cells. It was also found to protect glioma cells from radiotherapy-induced apoptosis. Co-immunoprecipitation (CO-IP) found that HSP90B1 directly interacted with RhoC and protected it from degradation via the ubiquitin–proteasome pathway. Rescue experiments indicated that HSP90B1 might facilitate glioma migration, invasion, and radiotherapy resistance by modulating RhoC expression. A mouse model further demonstrated that gliomas expressing high levels of HSP90B1 exhibited decreased sensitivity to radiotherapy. Overall, our research revealed that HSP90B1 significantly impacts the prognosis of glioma patients treated with radiotherapy. Additionally, HSP90B1 might enhance glioma metastasis and resistance to radiotherapy by regulating RhoC expression. This regulatory effect was achieved by the directly binding of HSP90B1 to RhoC, thereby preventing its degradation through the ubiquitin–proteasome pathway.