Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O 6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage.EPIC-1042 was obtained from Receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, Co-immunoprecipitation and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ.EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo.EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.