Granzyme B-induced apoptosis requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How granzyme B (GB), which does not possess a mitochondrial targeting sequence, enters this organelle is unknown. We show that GB enters mitochondria independently of the translocase of the outer mitochondrial membrane (TOM) complex but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane -barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat shock protein 70 (mtHsp70)-dependent manner. Granzyme A and caspase 3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated ROS and cell death. Therefore, cytotoxic molecules enter the mitochondria to efficiently induce cell death through a non-canonical Sam50, Tim22 and mtHsp70-dependent import pathway.