Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A2 (PLA2) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA2 enzymatic activity and how these critical residues contribute to its PLA2 activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca2+-dependent secreted PLA2-like (sPLA2) activity, which was inhibited by sPLA2-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA2 activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA2-like enzymatic activity, and these residues are crucial for its sPLA2-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.