Merkel cell polyomavirus (MCPyV) large tumor (LT) antigen is a DNA binding protein essential for viral gene transcription and genome replication. MCPyV LT interacts with multiple E3 ligases in a phosphorylation-dependent manner, limiting its own viral replication by enhancing LT protein degradation, which is a unique mechanism for MCPyV latency. Thus, identifying LT ubiquitination sites is an important step toward understanding the biological role of these virus-host interactions that can potentially result in viral oncogenesis. The ubiquitin (Ub) attachment sites in LT were predicted by using Rapid UBIquitination (RUBI), a sequence-based ubiquitination web server. Using an immunoprecipitation approach, the lysine (Lys, K) 585 residue in LT is identified as the ubiquitin conjugation site. Lysine 585 is deleted from tumor-derived truncated LTs (tLTs), resulting in stable expression of tLTs present in cancers. Substitution of lysine 585 to arginine (Arg, R) increased LT protein stability, but impaired MCPyV origin replication, due to a loss of ATP hydrolysis activity. These findings uncover a never-before-identified ubiquitination site of LT and its importance not only in the regulation of protein turnover, but also in MCPyV genome replication.