DNA damage contributes to renal tubular cell death during kidney injury, but how DNA damage in tubular cells is regulated is not fully understood. Lethal (3) malignant brain tumor-like 2 (L3MBTL2), a novel polycomb group protein, has been implicated in regulating chromatin architecture. However, the biological functions of L3MBTL2 are largely undefined. Here we found that L3MBTL2 was expressed in the nuclei of renal tubular epithelial cells in mice. Ablation of L3mbtl2 in renal tubular cells resulted in increases in nuclear DNA damage, p53 activation, apoptosis, tubular injury and kidney dysfunction after cisplatin treatment or unilateral ureteral obstruction. In vitro, inhibition of L3MBTL2 sequentially promoted histone γH2AX expression, p53 activation and apoptosis in cisplatin-treated mouse proximal tubular TKPTS cells. Inhibition of p53 activity attenuated the apoptosis induced by L3mbtl2 deficiency after cisplatin treatment both in vivo and in vitro. Intriguingly, unlike other polycomb proteins, L3MBTL2 was not recruited to DNA damage sites, but instead increased nuclear chromatin density and reduced initial DNA damage load. Thus, L3MBTL2 plays a protective role in kidney injury, in part by inhibiting the DNA damage-p53-apoptosis pathway.