Selective modulation of autophagy is a promising therapeutic strategy, especially for cancer treatment. However, the lack of specific autophagy inhibitors limits this strategy. The formation of the ATG12-ATG5-ATG16L1 complex is essential for targeting the ATG12-ATG5 conjugate to proper membranes and to generate LC3-II for the progression of autophagy. Thus, targeting ATG5-ATG16L1 protein-protein interactions (PPIs) might inhibit early stage autophagy with high specificity. In this paper, we report that a stapled peptide derived from ATG16L1 exhibits potent binding affinity to ATG5, striking resistance to proteolysis, and significant autophagy inhibition activities in cells.