Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer. The Src inhibitor, TAT-Cx43 _266-283 , exerts antitumor effects in in vitro and in vivo models of GBM. Because addressing the mechanism of action is essential to translate these results to a clinical setting, in this study we carried out an unbiased proteomic approach. Data-independent acquisition mass spectrometry proteomics allowed the identification of 190 proteins whose abundance was modified by TAT-Cx43 _266-283 . Our results were consistent with the inhibition of Src as the mechanism of action of TAT-Cx43 _266-283 and unveiled antitumor effectors, such as p120 catenin. Changes in the abundance of several proteins suggested that TAT-Cx43 _266-283 may also impact the brain microenvironment. Importantly, the proteins whose abundance was reduced by TAT-Cx43 _266-283 correlated with an improved GBM patient survival in clinical datasets and none of the proteins whose abundance was increased by TAT-Cx43 _266-283 correlated with shorter survival, supporting its use in clinical trials.