L-Ascorbic acid (vitamin C) in fruits and vegetables is an essential component of human nutrition. Surprisingly, only limited information is available about the pathway(s) leading to its biosynthesis in plants. Here, we report the isolation and characterization of GalUR, a gene from strawberry that encodes an NADPH-dependent D-galacturonate reductase. We provide evidence that the biosynthesis of L-ascorbic acid in strawberry fruit occurs through D-galacturonic acid, a principal component of cell wall pectins. Expression of GalUR correlated with changing ascorbic acid content in strawberry fruit during ripening and with variations in ascorbic acid content in fruit of different species of the genus Fragaria. Reduced pectin solubilization in cell walls of transgenic strawberry fruit with decreased expression of an endogenous pectate lyase gene resulted in lower ascorbic acid content. Overexpression of GalUR in Arabidopsis thaliana enhanced vitamin C content two- to threefold, demonstrating the feasibility of engineering increased vitamin C levels in plants using this gene.