Mesenchymal (MES) transition and tumor-associated macrophage (TAM) infiltration are two major events associated with poor prognosis during glioblastoma (GBM) progression. Here, transcriptome analysis of 48 longitudinal GBM primary and recurrent sample pairs revealed a meaningful association of MES subtype upon relapse with increased TAM recruitment. MES GBM-associated TAMs, but not non-MES GBM-associated TAMs, exhibited a unique capability to shift non-MES GBM cells toward the MES phenotype and enhance the tumor aggressiveness of non-MES GBM stem-like cells. A distinctive signature profile was defined for MES GBM-associated TAMs by transcriptome comparison among TAMs isolated directly from MES and non-MES tumors. The MES-TAM signature identified poor-outcome individuals independent of tumor subtypes at initial diagnosis and was predictive of MES differentiation during disease progression under therapy. Collectively, these data highlight the pivotal role of the interaction between GBM cells and TAMs in triggering the transcriptional MES transition in the course of GBM progression.