Human respiratory syncytial virus (hRSV) accounts for most lower respiratory tract infections during infancy and childhood. We previously demonstrated that the hRSV Matrix (M) protein interacts with the nucleolar protein Nucleophosmin 1 (NPM1) in HEK 293T cells. In this study we confirmed this interaction by using an in vitro pull-down assay. Knockdown of NPM1 with shRNA against NPM1 mRNA as well as the use of an NPM1 oligomerization inhibitor, affected hRSV proteins expression. These results indicate that the association of NPM1 with M is important for hRSV replication and is a possible target for antiviral strategies.