The P protein of parainfluenza virus 5 (PIV5) is an essential cofactor of the viral RNA-dependent RNA polymerase. Phosphorylation of the P protein can positively or negatively regulate viral gene expression, depending on the precise phosphorylation sites. Sumoylation, a process of adding small ubiquitin-like modifier (SUMO) to proteins posttranslationally, plays an important role in regulating protein function. In this study, we have found that the P protein of PIV5 was sumoylated with SUMO1 in both transfected and infected cells. The K254 residue of the P protein is within a consensus sumoylation motif. Mutation of the P protein at K254 to arginine (P-K254R) reduced PIV5 minigenome activity, as well as the sumoylation level of the P protein. Incorporation of K254R into a recombinant PIV5 (rPIV5-P-K254R) resulted in a virus that grew to a lower titer and had lower levels of viral RNA synthesis and protein expression than wild-type PIV5, suggesting that sumoylation of the P protein at K254 is important for PIV5 growth. Biochemical studies did not reveal any defect of P-K254R in its interactions with viral proteins NP and L or formation of homotetramers. We propose that sumoylation of the P protein at K254 regulates PIV5 gene expression through a host protein.