Infection of plum bark necrosis stem pitting associated virus (PBNSPaV) has been reported in many prunus species in several countries, causing significant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes in cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, a PVX-P6 expression vector was constructed by inserting the p6 fragment into a potato virus X (PVX)-based vector and transformed into Agrobacterium tumefaciens GV3101. Infiltration of N. benthamiana with the PVX vector-transformed A. tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, infiltration with the PVX-P6 vector-transformed A. tumefaciens resulted in no significant symptoms. These results demonstrated that heterologous expression of P6 in N. benthamiana could not enhance the pathogenicity of PVX. Our study indicates that p6 may not be a potential pathogenic factor associate with the causing of symptoms, and mode of action of PBNSPaV-P6 protein remain to be further studied.