Vasculogenic mimicry (VM) describes the unique ability of highly aggressive melanoma tumor cells to express endothelial cell-associated genes (such as EphA2 and VE-cadherin) and form vasculogenic-like networks when cultured on a three-dimensional matrix. VM has been described in several types of aggressive tumors, including melanoma, prostate, breast, and ovarian carcinomas. However, the molecular underpinnings of this phenomenon remain somewhat elusive. In this study, we examined key molecular mechanisms underlying VM in aggressive human cutaneous and uveal melanoma. The data reveal that phosphoinositide 3-kinase (PI3K) is an important regulator of VM, specifically affecting membrane type 1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) activity, critical in the formation of vasculogenic-like networks. Using specific inhibitors of PI3K, melanoma VM was abrogated coincident with decreased MMP-2 and MT1-MMP activity. Furthermore, inhibition of PI3K blocked the cleavage of laminin 5 gamma 2 chain, resulting in decreased levels of the gamma 2' and gamma 2x promigratory fragments. Collectively, these results indicate that PI3K is an important regulator of melanoma VM directly affecting the cooperative interactions of MMP-2, MT1-MMP, and laminin 5 gamma 2 chain and, thus, the remodeling of the tumor cell microenvironment. PI3K may represent an excellent target for therapeutic intervention of a novel signaling cascade underlying VM.