D-Pantolactone is a key chiral intermediate for the synthesis of D-pantothenic acid and its derivatives. Biocatalytic kinetic resolution of D,L-pantoyl lactone using D-lactonase is an efficient route to synthesize D-pantolactone. In this study, we report the expression of a novel D-lactonase TSDL in Escherichia coli host. The recombinant TSDL exhibited high hydrolysis activity and enantioselectivity toward D-pantolactone. The reaction conditions of the recombinant TSDL-catalyzed kinetic resolution of D,L-pantolactone was systematically investigated by whole cell biocatalysis. In addition, a preparative-scale reaction for bioproduction of D-pantoic acid was examined under optimized reaction conditions. This study presented an alternative enzymatic process for kinetic resolution of D,L-pantolactone.