AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. This study reveals that AMPK activates Ulk1 by phosphorylating Ser 317 and Ser 777, while mTORC1 inhibits Ulk1 by phosphorylating Ser 757, thereby regulating autophagy. Under glucose starvation, AMPK promotes autophagy by activating Ulk1 through phosphorylation, whereas mTORC1 prevents Ulk1 activation by phosphorylating Ser 757 and disrupting the interaction between Ulk1 and AMPK. These phosphorylation events are crucial for Ulk1 function in autophagy induction. The study also shows that AMPK is essential for Ulk1 activation in response to glucose starvation, while mTORC1 activity inhibits this process. The findings highlight the coordinated regulation of autophagy by AMPK and mTORC1 through phosphorylation of Ulk1, providing insights into the molecular mechanisms underlying autophagy regulation in response to nutrient signals.AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. This study reveals that AMPK activates Ulk1 by phosphorylating Ser 317 and Ser 777, while mTORC1 inhibits Ulk1 by phosphorylating Ser 757, thereby regulating autophagy. Under glucose starvation, AMPK promotes autophagy by activating Ulk1 through phosphorylation, whereas mTORC1 prevents Ulk1 activation by phosphorylating Ser 757 and disrupting the interaction between Ulk1 and AMPK. These phosphorylation events are crucial for Ulk1 function in autophagy induction. The study also shows that AMPK is essential for Ulk1 activation in response to glucose starvation, while mTORC1 activity inhibits this process. The findings highlight the coordinated regulation of autophagy by AMPK and mTORC1 through phosphorylation of Ulk1, providing insights into the molecular mechanisms underlying autophagy regulation in response to nutrient signals.