As a key immune checkpoint ligand, PD-L1 is a critical target in cancer immunotherapy. While multiple E3 ubiquitin ligases including CRL3(SPOP), ARIH1, and NEDD4 have been implicated in PD-L1 degradation, the precise enzymatic mechanisms remain unclear. In this study, we systematically compared the enzymatic activities of CRL3(SPOP), ARIH1, and NEDD4 ligases toward the cytoplasmic domain of PD-L1 through in vitro reconstitution with purified components. ARIH1, rather than CRL3(SPOP), independently ubiquitinates PD-L1. We reveal a mechanism where ARIH1 acts as a substrate receptor and cooperates with CRLs to catalyze PD-L1 ubiquitination. We also biochemically validated the E3 ligase activity of the NEDD4 family E3s toward PD-L1. By using liposomes in the enzymatic assays, we show that phosphorylation enhances PD-L1 ubiquitination through disrupting its membrane association. Our study provides further biochemical insights into PD-L1 ubiquitination, which advances our understanding of the molecular details of PD-L1 regulation.