As aldehyde dehydrogenase (ALDH) is a novel stem cell marker, increasing studies have confirmed that high ALDH activity promotes tumorigenesis and progression in cancers. Some preliminary studies have found that ALDH1A3 may play an important role in glioma malignant progression, but so far there was no conclusive conclusion. The purpose of our study was to elucidate the mechanisms by which ALDH1A3 regulated in glioma and to provide practical tools for clinical application. Aldefluor, flow cytometry sorting and qRT-PCR were performed to verify the role of ALDH1A3 in ALDH activity maintenance. Transwell, immunofluorescence, glycolytic assays, and orthotopic xenograft models were used to explore ALDH1A3 bio-functions in GBM. LASSO-COX, COX survival analysis and Kaplan-Meier analysis were used to establish the prognostic evaluation system and predict postoperative chemotherapy sensitivity of GBMs. Our integrated study found that (1) ALDH1A3 associates with mesenchymal differentiation of GBM in Eastern and Western world patients. (2) ALDH1A3 plays a critical role in ALDH activity maintenance. (3) ALDH1A3 is an activator of mesenchymal transformation in GBM. (4) ALDH1A3-derived PMT markers' molecular signature can predict 1-, 2-, and 3-year survival rates of GBMs precisely. In conclusion, ALDH1A3 was a major contributor to ALDH activity and a key driver in triggering mesenchymal transformation in GBM. ALDH1A3-based molecular classification scheme can help to improve guidance for prognosis forecasting and individualized treatment decision making for GBM patients.