UDP-sugar pyrophosphorylase catalyzes the conversion of various monosaccharide 1-phosphates to the respective UDP-sugars in the salvage pathway. Using the genomic database, we cloned a putative gene for UDP-sugar pyrophosphorylase from Arabidopsis. Although relatively stronger expression was detected in the vascular tissue of leaves and the pollen, AtUSP is expressed in most cell types of Arabidopsis, indicating a housekeeping function in nucleotide sugar metabolism. Recombinant AtUSP expressed in Escherichia coli exhibited broad specificity toward monosaccharide 1-phosphates, resulting in the formation of various UDP-sugars such as UDP-glucose, -galactose, -glucuronic acid, -xylose and -L-arabinose. A loss-of-function mutation in the AtUSP gene caused by T-DNA insertion completely abolished male fertility. These results indicate that AtUSP functions as a UDP-sugar pyrophosphorylase in the salvage pathway, and that the generation of UDP-sugars from monosaccharide 1-phosphates catalyzed by AtUSP is essential for pollen development in Arabidopsis.