The main protease (Mpro, also known as 3CLpro) of SARS-CoV-2 plays a pivotal role in the viral life cycle by mediating the cleavage of viral polyproteins. Understanding the regulatory mechanisms of Mpro, including its protein-protein interactions (PPIs) and post-translational modifications (PTMs), is crucial for developing therapeutic strategies. In this study, we employed high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to systematically investigate the Mpro interactome and its modification status. To identify potential E3 ubiquitin ligases that regulate Mpro stability or function, we performed immunoprecipitation-mass spectrometry (IP-MS) using 293T cells expressing Flag-tagged Mpro. Additionally, we mapped the phosphorylation sites of Mpro to elucidate how host-cell kinases modulate its enzymatic activity. Our data revealed several key E3 ligases that interact with Mpro and identified novel phosphorylation sites on the protease. This dataset provides a comprehensive resource for understanding the host-virus interaction and the precise regulation of SARS-CoV-2 replication.