Glutarate is an important C5 linear chain dicarboxylic acid having wide applications in chemical industry. In this work we report glutarate production by Escherichia coil with a newly constructed biosynthetic pathway containing part of known glutaconate biosynthetic pathway and a gap-filling module employing trans-enoyl-CoA reductase (Ter). Overall the artificial pathway comprises reduction of the central carbon metabolite a-ketoglutarate to 2-hydroxyglutarate, activation to 2-hydroxyglutaryl-CoA, dehydration to trans-glutaconyl-CoA, hydrogenation to glutaryl-CoA by Ter and thioester hydrolysis to finally yield glutarate. The pathway introduced into E. coil resulted in a recombinant strain that produced 3.8 mg/L of glutarate together with 27.7 mg/L of glutaconate in anaerobic culture mode. The glutarate production increased by approximately 50% through the mutation of Ter from Treponema denticola. The results demonstrated biosynthesis of glutarate via a non-natural synthetic pathway, which may enable its biobased production from renewable resources.