unc‐51‐like autophagy activating kinase 1 and 2 (Ulk1/2) regulate autophagy initiation under various stress conditions. However, the physiological functions of these Ser/Thr kinases are not well characterized. Here, we show that mice with liver‐specific double knockout (LDKO) of Ulk1 and Ulk2 (Ulk1/2 LDKO) are viable, but exhibit overt hepatomegaly phenotype. Surprisingly, Ulk1/2 LDKO mice display normal autophagic activity in hepatocytes upon overnight fasting, but are strongly resistant to acetaminophen (APAP)‐induced liver injury. Further studies revealed that Ulk1/2 are also dispensable for APAP‐induced autophagy process, but are essential for the maximum activation of c‐Jun N‐terminal kinase (JNK) signaling both in vivo and in isolated primary hepatocytes during APAP treatment. Mechanistically, APAP‐induced inhibition of mechanistic target of rapamycin complex 1 releases Ulk1 from an inactive state. Activated Ulk1 then directly phosphorylates and increases the kinase activity of mitogen‐activated protein kinase kinase 4 and 7 (MKK4/7), the upstream kinases and activator of JNK, and mediates APAP‐induced liver injury. Ulk1‐dependent phosphorylation of MKK7 was further confirmed by a context‐dependent phosphorylation antibody. Moreover, activation of JNK and APAP‐induced cell death was markedly attenuated in Mkk4/7 double knockdown hepatocytes reconstituted with an Ulk1‐unphosphorylatable mutant of MKK7 compared to those in cells rescued with wild‐type MKK7. Conclusion: Together, these findings reveal an important role of Ulk1/2 for APAP‐induced JNK activation and liver injury, and understanding of this regulatory mechanism may offer us new strategies for prevention and treatment of human APAP hepatotoxicity. (Hepatology 2018;67:2397‐2413).