The interactions between host cellular proteins and viral proteins are important for successful infection by viruses. Previous studies from our group have identified various host cellular proteins that can interact with the Newcastle disease virus V protein (Chu et al., 2018a), but their function in NDV replication has not been fully determined. The present study reports that heterogenous nuclear ribonucleoprotein H1 (hnRNP H1) can interact with NDV V protein in yeast. The immunofluorescence results showed that hnRNP H1 and V protein could colocalize in the cytoplasm of a chicken embryo fibroblast cell line (DF-1 cells). Co-immunoprecipitation assays further verified the interaction of these two proteins. The effects of overexpression and knockdown of hnRNP H1 on NDV replication were evaluated in DF-1 cells through real time quantitative PCR (RT-qPCR) and plaque assays. The regulation of V protein on hnRNP H1 expression was also examined. The results indicated that overexpression of hnRNP H1 facilitated NDV replication, while knockdown of hnRNP H1 decreased NDV replication. It was also shown that V protein could regulate hnRNP H1 expression at the protein level instead of the transcription level. The effect of V protein and hnRNP H1 on the DF-1 cell cycle was also tested and the results revealed that V protein may regulate cell proliferation by controlling the expression of hnRNP H1. Taken together, these results suggest that NDV V protein could promote viral replication by interacting with hnRNP H1.