Vasculogenic mimicry (VM) refers to the process by which highly aggressive tumor cells mimic endothelial cells to form vessel-like structures that aid in supplying enough nutrients to rapidly growing tumors. Histone deacetylases (HDACs) regulate the expression and activity of numerous molecules involved in cancer initiation and progression. Notably, HDAC3 is overexpressed in the majority of carcinomas. However, thus far, no data are available to support the role of HDAC3 in VM. In this study, we subjected glioma specimens to immunohistochemical and histochemical double-staining methods and found that VM and HDAC3 expression were related to the pathological grade of gliomas. The presence of VM correlated with HDAC3 expression in glioma tissues. The formation of tubular structures, as determined by the tube formation assay to evaluate VM, was impaired in U87MG cells when transfected by siRNA or treated with an HDAC3 inhibitor. Importantly, the expression of VM-related molecules such as MMP-2/14 and laminin5γ2 was also affected when HDAC3 expression was altered. Furthermore, U87MG cells were treated with a phosphoinositide 3-kinase (PI3K) inhibitor or/and ERK inhibitor and found that the PI3K and ERK signaling pathways play key roles in VM; whereas, in VM, the two signaling pathways did not act upstream or downstream from each other. Taken together, our findings showed that HDAC3 contributed to VM in gliomas, possibly through the PI3K/ERK-MMPs-laminin5γ2 signaling pathway, which could potentially be a novel therapeutic target for gliomas. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.