The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasion-promoting role of microglia are unknown. Transforming growth factor-beta (TGF-beta) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-beta activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-beta signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-beta type II receptor (TbetaIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbetaIIR abolished TGF-beta-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbetaIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbetaRII, indicating a crucial role of microglia-derived TGF-beta in tumor-host interactions. Our results demonstrate a successful targeting of TGF-beta-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.