||Aleutian mink disease virus (AMDV) is the only member in genus Amdovirus of the family Parvoviridae. During AMDV infection, six species of viral transcripts are generated from one precursor mRNA through alternative splicing and alternative polyadenylation. In addition to the large non-structural protein NS1, two small non-structural proteins, NS2 and NS3, are putatively encoded (Qiu J, et al., 2006. J. Virol. 80 654-662). However, these two proteins have not been experimentally demonstrated during virus infection, and nothing is known about their function. Here, we studied the nonstructural protein expression profile of AMDV, and for the first time, confirmed expression of NS2 and NS3 during infection, and identified their intracellular localization. More importantly, we provided evidence that both NS2 and NS3 are necessary for AMDV replication.