BackgroundFor cancer therapy, the identification of both selective autophagy targets and small molecules that specifically regulate autophagy is greatly needed. Heat shock protein 70 (Hsp70) is a recently discovered BH3 receptor that forms a protein‒protein interaction (PPI) with Bcl-2-interacting mediator of cell death (Bim). Herein, a specific inhibitor of the Hsp70-Bim PPI,S1g-2, and its analogS1, which is a Bcl-2-Bim disruptor, were used as chemical tools to explore the role of Hsp70-Bim PPI in regulating mitophagy.MethodsCo-immunoprecipitation and immunofluorescence assays were used to determine protein interactions and colocalization patterns. Organelle purification and immunodetection of LC3-II/LC3-I on mitochondria, endoplasmic reticulum (ER) and Golgi were applied to identify specific types of autophagy. Cell-based and in vitro ubiquitination studies were used to study the role of the Hsp70-Bim PPI in parkin-mediated ubiquitination of outer mitochondrial membrane 20 (TOMM20).ResultsWe found that after the establishment of their PPI, Hsp70 and Bim form a complex with parkin and TOMM20, which in turn facilitates parkin translocation to mitochondria, TOMM20 ubiquitination and mitophagic flux independent of Bax/Bak. Moreover,S1g-2selectively inhibits stress-induced mitophagy without interfering with basal autophagy.ConclusionsThe findings highlight the dual protective function of the Hsp70-Bim PPI in regulating both mitophagy and apoptosis.S1g-2is thus a newly discovered antitumor drug candidate that drives both mitophagy and cell death via apoptosis.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s11658-023-00458-5.