AlkB homolog 5, RNA demethylase (ALKBH5) is abnormally highly expressed in glioblastoma multiforme (GBM) and is negatively correlated with overall survival in GBM patients. In this study, we found a new mechanism that ALKBH5 and pyrroline-5-carboxylate reductase 2 (PYCR2) formed a positive feedback loop involved in proline synthesis in GBM. ALKBH5 promoted PYCR2 expression and PYCR2-mediated proline synthesis; while PYCR2 promoted ALKBH5 expression through the AMPK/mTOR pathway in GBM cells. In addition, ALKBH5 and PYCR2 promoted GBM cell proliferation, migration, and invasion, as well as proneural-mesenchymal transition (PMT). Furthermore, proline rescued AMPK/mTOR activation and PMT after silencing PYCR2 expression. Our findings reveal an ALKBH5-PYCR2 axis linked to proline metabolism, which plays an important role in promoting PMT in GBM cells and may be a promising therapeutic pathway for GBM.