Non-collagenous α3 chain of type IV collagen or α3(IV)NC1, a 28 kDa C-terminal domain of collagen type IV is a specific inhibitor of endothelial cell translation and angiogenesis. In the present study we have cloned and expressed mouse α3(IV)NC1 in baculovirus system. The recombinant protein was expressed in soluble form and tested for several of its biological functions. We identified that this recombinant mouse α3(IV)NC1 specifically inhibited proliferation, translation and tube formation of endothelial cells. Also, we show that α3(IV)NC1 treatment results in apoptosis specifically in proliferating endothelial cells. In addition we report for the first time that mouse α3(IV)NC1 inhibits migration and p38 MAPK phosphorylation in addition to inhibition of FAK/Akt/mTOR/4E-BP1 signaling. In mice α3(IV)NC1 treatment reduced tumor growth and CD-31 positive endothelial vasculature in tumors. Collectively, our data demonstrate the expression of biologically active form of mouse α3(IV)NC1 in Sf-9 cells and provide important mechanistic insights on α3(IV)NC1 antiangiogenic actions in endothelial cells.