This study combined co-immunoprecipitation (Co-IP) and LC-MS/MS proteomics in MARCH8-overexpressing HeLa cells to identify potential substrates of the E3 ubiquitin ligase MARCH8. Using empty vector-transfected cells as controls, we generated a comprehensive protein interaction dataset to elucidate MARCH8’s regulatory mechanisms in membrane protein trafficking and degradation, providing a resource for exploring its roles in viral defense, immune modulation, and related pathologies.