BK virus (BKV) infection may cause polyomavirus-associated nephropathy in patients with renal transplantation. Recently, the phosphorylated amino acids on the structural proteins VP1, VP2 and VP3 of BKV have been identified by liquid chromatography-tandem mass spectrometry in our laboratory. In this study, we further analysed the biological effects of these phosphorylation events. Phosphorylation of the BKV structural proteins was demonstrated by [(32)P]orthophosphate labelling in vivo. Site-directed mutagenesis was performed to replace all of the phosphorylated amino acids. The mutated BKV genomes were transfected into Vero cells for propagation analysis. The results showed that expression of the early protein LT and of the late protein VP1 by the mutants VP1-S80A, VP1-S80-133A, VP1-S80-327A, VP1-S80-133-327A and VP2-S254A was abolished. However, propagation of other mutants was similar to that of wild-type BKV. The results suggest that phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is crucial for BKV propagation.