Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus onMprotein and identify a key phosphorylation site (Thr205) inMthat is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation inM(serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affectMstability or the ability to form dimers but implicate an effect on higherorder oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impairMoligomerization; typical filamentous structures still formed at the plasma membrane, butMassembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time thatMoligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV.