Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles.Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear.In this work, one novel chemical, G1, was screened out which could trigger both canonical autophagy and noncanonical autophagy by recruiting Atg16L1 to preautophagosomal site and causing LC3 lipidation.The G1induced noncanonical autophagy was ULK1, and Beclin1independent but ubiquitinlike conjugation systemdependent, indicating G1 might target the upstream of Atg16L1.Moreover, inhibition of VATPase by specific VATPase inhibitiors could suppress the formation of G1induced autophagosomes in FIP200deficient MEF cells.While other classic lysosomal inhibitors could not block the puncta of Atg12, Atg16L1 and LC3, in different stages, suggesting VATPase activity instead of lysosome function is required for G1induced noncanonical autophagy.These studies broaden the understanding of different working pattern of autophagy and the crucial roles of VATPase in the regulation of different autophagy.