Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD). To date, effective treatments for VC are still lacking since the underlying mechanisms remains elusive. RNA-sequencing analysis revealed that the E3 ubiquitin ligase Tripartite motif 16 (TRIM16) may be a key modulator of VC. However, it remains unclear whether TRIM16-mediated ubiquitination contributes to VC. In the present study, we used LC-MS/MS to identify the substrates of TRIM16.