Intersectin1 (ITSN1) contains two isoforms: ITSN1-S and ITSN1-L, which is highly regulated by alternative splicing. However, the alteration of alternative splicing and its importance in cancer is still unknown. In this study, our transcriptome analysis by using a large glioma cohort indicated the two isoforms exerted opposite function in glioma progression. Our previous results had shown ITSN1-S could promote glioma development; however, the function of ITSN1-L remained unknown. In this study, we first confirmed that ITSN1-L exerted an inhibitory role in glioma progression both in vivo and in vitro, which was contrary to the function of ITSN1-S. In additional, we also elucidated the mechanisms of ITSN1-L in inhibiting tumor progression. First, we revealed ITSN1-L could interact with α-tubulin to promote HDAC6-dependent deacetylation of ac-tubulin leading to decreased cell motility. Second, ITSN1-L could attenuate cell-substrate adhesion through FAK/integrin β3 pathway. Third, ITSN1-L was able to strengthen cell-cell adhesion by upregulating N-cadherin expression and its re-localization to membrane by ANXA2 and TUBB3/TUBB4. In conclusion, we found for the first time that two isoforms produced by alternative splicing exerted opposite functions in glioma development. Therefore, upregulation of ITSN1-L expression as well as downregulation of ITSN1-S expression probably was a better strategy in glioma treatment. Our present study laid a foundation for the importance of alternative splicing in glioma progression and raised the possibility of controlling glioma development completely at an alternative splicing level to be a more effective strategy.