Ubiquitin linkage is critical in directing the cellular fate of a ubiquitinated protein.Although K48-1inked polyubiquitination of p53 leads to its degradation, whether K48-independent ubiquitin linkages are involved in p53 activation remains unknown.Here, we show that FATS acts as a p53 activator through inhibiting Mdm2 binding to p53 and stimulating non-proteolytic polyubiquitination of p53.Knockdown of FATS impairs p53 stabilization and activation in response to DNA damage.Furthermore, the NH2-terminal domain of FATS is sufficient to exhibit ubiquitin ligase (E3) activity and assemble ubiquitin polymers through K11-, K29-and K63-1inkages, independently of ubiquitin-conjugating enzyme (E2).FATS promotes p53-dependent transcription of p21, leading to robust checkpoint response.An E3-defective mutant of FATS fails to promote p53 activation after DNA damage.Our findings reveal K63-1inked non-linear polyubiquitination of p53 as a new barcode for p53 activation.