BACKGROUND: Glioblastoma (GBM) is one of the most malignant types of central nervous system tumors. Oxygen deprivation in the tumor microenvironment is thought to be an important factor in promoting GBM progression. However, the mechanisms of hypoxia-promoted tumor progression remain elusive. METHODS: Alternative splicing of diacylglycerol kinase gamma (DGKG)-Δ exon13 was amplified and verified by PCR-Sanger sequencing. The functions of DGKG and DGKG-Δ exon13 were analyzed by Cell counting kit-8 (CCK-8), Transwell, Matrigel-transwell experiments, and in vivo orthotropic GBM animal models. Transcriptome analyses were done to find out the regulated genes. RESULTS: In this study, we found that a new transcript DGKG-Δ exon13 was generated in GBM under hypoxia via alternative splicing. Moreover, the results of CCK-8, Transwell, and Matrigel-transwell experiments showed that the proliferation, migration, and invasion abilities of U87-MG and T98G were decreased after DGKG knockdown. Compared to wild-type DGKG, DGKG-Δ exon13 overexpression significantly promoted cellular proliferation, migration, and invasion abilities in GBM. Furthermore, in vivo, orthotropic GBM animal models analysis showed that the tumor volumes were much smaller in the DGKG knockdown group. However, the tumor sizes in the DGKG and DGKG-Δ exon13 rescue groups were restored, especially in the DGKG-Δ exon13 group. Transcriptome analysis revealed that MORC1, KLHDC7B, ATP1A2, INHBE, TMEM119, and FGD3 were altered significantly when DGKG was knocked down. IL-16, CCN2, and EFNB3 were specifically regulated by DGKG-Δ exon13. CONCLUSIONS: Our study found that hypoxia-induced alternative splicing transcript DGKG-Δ exon13 promotes GBM proliferation and infiltration, which might provide a new potential target for the clinical treatment and diagnosis of GBM.