Forkhead transcription factor FoxO3a has been reported to have ambiguous functions and distinct mechanisms in various solid tumors, including glioblastoma (GBM). Although a preliminary analysis of a small sample of patients indicated that FoxO3a aberrations in glioma might be related to aggressive clinical behavior, the clinical significance of FoxO3a in glioblastoma remains unclear. We investigated the expression of FoxO3a in a cohort of 91 glioblastoma specimens and analyzed the correlations of protein expression with patient prognosis. Furthermore, the functional impact of FoxO3a on GBM progression and the underlying mechanisms of FoxO3a regulation were explored in a series of in vitro and in vivo assays. FoxO3a expression was elevated in glioblastoma tissues, and high nuclear FoxO3a expression in human GBM tissues was associated with poor prognosis. Moreover, knockdown of FoxO3a significantly reduced the colony formation and invasion ability of GBM cells, whereas overexpression of FoxO3a promoted the colony formation and invasion ability. The results of in vivo GBM models further confirmed that FoxO3a knockdown inhibited GBM progression. More, the pro-oncogenic effects of FoxO3a in GBM were mediated by the activation of c-Myc, microtubule-associated protein 1 light chain 3 beta (LC3B) and Beclin1 in a mixed-lineage leukemia 2 (MLL2)-dependent manner. These findings suggest that high FoxO3a expression is associated with glioblastoma progression and that FoxO3a independently indicates poor prognosis in patients. FoxO3a might be a novel prognostic biomarker or a potential therapeutic target in glioblastoma. What's new? Genetic aberrations in forkhead transcription factor (FoxO3a) are thought to play a role in aggressive glioblastoma (GBM), though the nature of their involvement remains unclear. In this study, high FoxO3a expression was positively correlated with GBM progression and poor prognosis in patients. In cells, FoxO3a was found to promote GBM progression via activation of c-Myc and the autophagy-related proteins LC3B and Beclin1. FoxO3a pro-oncogenic effects were mediated by the epigenetic factor MLL2. In addition, in mice, FoxO3a knockdown inhibited GBM progression. The findings suggest that FoxO3a is a potential prognostic biomarker or therapeutic target in GBM.