AMPK and mTOR play crucial roles in regulating cellular metabolism and cell proliferation. Dysregulation of these pathways is associated with various diseases, including cancer, type 2 diabetes, and neurodegenerative disorders. Our recent study revealed a previously unknown bidirectional regulation between AMPK and the mTORC1 complex. We found that mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient-stress conditions. Specifically, mTORC1 phosphorylates the catalytic subunit of AMPKα2 at Ser345, leading to decreased AMPK activity. Furthermore, we demonstrated that the dephosphorylation of α2-S345 and/or inhibition of mTORC1 temporarily directs AMPK to the lysosome for activation by LKB1. To identify the cellular substrates of AMPK during transient translocation to the lysosome, we conducted a lysosome-enriched label-free phosphoproteomics analysis. Lentivirus was employed to introduce wild-type AMPK (AMPKα2WT) and kinase-dead AMPK (AMPKα2KD) into AMPKα1/2 dKO iMEFs stably transfected with 3×HA-TMEM192, enabling rapid isolation of intact lysosomes using the Lyso-IP technique. Following Torin-1 treatment for 1 hour to inhibit mTORC1, we isolated lysosome-enriched fractions and performed label-free global proteomics and phosphoproteomics analyses on these fractions. Our analysis yielded over 3300 proteins and 2050 phosphopeptides from the lysosome-enriched fractions. A significant increase in phosphorylation was observed in SLC4A7, TAGLN2, BUD13, STX17, CDC42BPA, and other substrates when comparing AMPKα2WT with AMPKα2KD. Conversely, RASA3 displayed a substantial decrease in phosphorylation, and reduced protein level expression of CREG1 and NUBP2. We have validated SLC4A7 and TAGLN2 as novel AMPK substrates. Our study indicates the involvement of AMPK in a vast array of still unexplored cellular functions.