The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome and is activated by the E1 enzyme UBA6. In this study, we identify the UBA6-specific E2 enzyme (USE1) as an interaction partner of FAT10. Activated FAT10 can be transferred from UBA6 onto USE1 in vitro, and endogenous USE1 and FAT10 can be coimmunoprecipitated from intact cells. Small interfering RNA-mediated downregulation of USE1 mRNA resulted in a strong reduction of FAT10 conjugate formation under endogenous conditions, suggesting that USE1 is a major E2 enzyme in the FAT10 conjugation cascade. Interestingly, USE1 is not only the first E2 enzyme but also the first known substrate of FAT10 conjugation, as it was efficiently auto-FAT10ylated in cis but not in trans.