The immunoprecipitation combined with mass spectrometry (IP-MS) technique was employed to systematically identify the host proteins that physically interact with the RSV M2-1 protein. To achieve reliable detection, the M2-1 protein with a Myc tag was transiently overexpressed in human embryonic kidney 293T cells, and then anti-tag immunoprecipitation was performed under natural conditions. Subsequently, the host proteins co-precipitated were identified and quantified using high-resolution liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This unbiased proteomic screening revealed the comprehensive interactions between M2-1 and host factors. These findings provided mechanistic insights into the directed regulation of RSV replication by the host and pointed out potential host-dependent factors for therapeutic intervention against RSV infection.