A number of chemicals with high industrial value can be synthesized from levulinic acid, a feasible building block readily available from cellulosic biomass. Among them, gamma-valerolactone is a versatile chemical precursor for the synthesis of value-added products including bio-active molecules, bio-fuels, and carbon-based chemicals. In this study, a novel two-step chemoenzymatic conversion of levulinic acid to (R)-gamma-valerolactone via 4-hydroxyvaleric acid was investigated. For that purpose, an engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) with improved catalytic activity toward levulinic acid was employed in the first-step reaction, and dehydration with 1 % (v/v) sulfuric acid was applied for the lactonization of 4-hydroxyvaleric acid to gamma-valerolactone in the second step. As a result, enantiomerically pure (R)-gamma-valerolactone ( > 99 % ee) was successfully produced from the free acid form of levulinic acid with the maximum yield of approximately 100 %.