Glioma invasiveness is accomplished in part by matrix metalloproteinases (MMPs) which remodel the constraints of the three dimensional (3D) matrix of the brain parenchyma. Tissue culture studies have advanced knowledge of glioma invasiveness but the majority of studies have used a two dimensional (2D) monolayer culture system which does not reproduce the spatial constraints of invasiveness in vivo. Here, we have used a 3D matrix of type I collagen (CL) gel to address glioma invasiveness in vitro. We show that in 3D CL matrix, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), cytokines which are elevated in gliomas in vivo, increased glioma cell invasiveness with correspondent elevation of MMP-2 and MMP-9. Cytokine-stimulated glioma invasiveness was blocked by three pharmacological metalloproteinase inhibitors and by small interfering RNAs to MMP-2. Thus, in 3D matrix of CL, MMP-2 expression is modulated by inflammatory cytokines with the concomitant increase in glioma invasiveness.