Glioblastoma multiforme (GBM), whose pathogenesis involves proneural-to-mesenchymal transition (PMT), is the most malignant type of glioma and is associated with a bleak prognosis. Lactate dehydrogenase (LDH) comprises two major subunits, LDHA and LDHB, which can assemble into five different isoenzymes (LDH1-5). However, the role of LDH isoenzyme and its subunits in different GBM subtypes is largely unknown. Our findings reveal that LDHA and LDHB subunits correlated with mesenchymal and proneural subtype classification, and have prognostic and clinical significance in GBM patients. Moreover, it is demonstrated that LDH5, characterized by high LDHA and low LDHB levels, is highly expressed in mesenchymal subtype GBM cells and promotes proliferation, migration, and PMT. Conversely, proneural subtype GBM cells exhibited LDH1 dominance, and low LDHA and high LDHB levels. Notably, LDH1 played a pivotal role in the proliferation, migration, and PMT of proneural glioma cells. For treatment of proneural subtype GBM, gossypol-acetic acid (GAA)-bovine serum albumin (BSA) nanoparticles (GAA-BSA NPs) were developed to ameliorate PMT by targeting LDH1. These nanoparticles effectively suppress proneural subtype tumor growth both and , surpassing their efficacy against the mesenchymal subtype. The results offer several novel insights into the role of LDH isoenzyme in subtype classification between mesenchymal and proneural GBM and provide a promising therapeutic approach for proneural subtype GBM.