Snakehead vesiculovirus (SHVV) is a kind of fish rhabdovirus that has caused great economic losses in snakehead fish culture in China. However, the mechanism on SHVV’s pathogenicity is still unclear. The phosphoprotein (P) isomers have been identified in mammalian rhabdovirus rabies virus and played important roles in the proliferation of rabies virus, but the function of P isomers in fish rhabdovirus has not been investigated. In order to identify the SHVV P isomers and study their roles in virus proliferation, the P gene of SHVV was amplified, and a prokaryotic expression plasmid pET32a-P was constructed. The recombinant His-P protein was purified by Ni-NTA affinity chromatography column, and polyclonal antibody against the His-P protein was acquired through immunization of New Zealand white rabbits. Using the P protein polyclonal antibody, the SHVV P isomers were determined. Furthermore, the subcellular localization of P isomers was assessed using enhanced green fluorescent protein (EGFP), and the effect of overexpression of P isomers on SHVV proliferation was determined using qRT-PCR, Western Blot, and TCID₅₀. The results showed that three P protein bands were detected during SHVV-infected channel catfish ovary (CCO) cells. Further verification determined that two of the three bands were P protein (also called P1) and its isomer P2. Subcellular localization experiment showed that both P1 and P2 localized mainly in cytoplasm, and P1 and P2 could shuttle between nucleus and cytoplasm. Overexpression of P1 or P2 in CCO cells increased G mRNA level about four times and G protein level around two times. The viral titer of SHVV was also significantly promoted via overexpression of P1 and P2, indicating that P1 and P2 promoted SHVV proliferation. Overall, SHVV can produce P protein (P1) and its isomer P2 during its infection, and P1 and P2 play an important role in SHVV proliferation. Our results will be helpful to understand SHVV pathogenicity and the function of P isomers of rhabdoviruses.