The mitochondrial unfolded protein response (UPR^mt) is a cytoprotective signaling pathway triggered by mitochondrial dysfunction. UPR^mt activation upregulates chaperones, proteases, antioxidants, and glycolysis at the gene level to restore proteostasis and cell energetics. Activating transcription factor 5 (ATF5) is a proposed mediator of the mammalian UPR^mt. Herein, we hypothesized pharmacological UPR^mt activation may protect against cardiac ischemia-reperfusion (I/R) injury in an ATF5-dependent manner. Accordingly, in vivo administration of the UPR^mt inducers oligomycin or doxycycline 6 h before ex vivo I/R injury (perfused heart) was cardioprotective in wild-type but not global Atf5^−/− mice. Acute ex vivo UPR^mt activation was not cardioprotective, and loss of ATF5 did not impact baseline I/R injury without UPR^mt induction. In vivo UPR^mt induction significantly upregulated many known UPR^mt-linked genes (cardiac quantitative PCR and Western blot analysis), and RNA-Seq revealed an UPR^mt-induced ATF5-dependent gene set, which may contribute to cardioprotection. This is the first in vivo proof of a role for ATF5 in the mammalian UPR^mt and the first demonstration that UPR^mt is a cardioprotective drug target. NEW & NOTEWORTHY Cardioprotection can be induced by drugs that activate the mitochondrial unfolded protein response (UPR^mt). UPR^mt protection is dependent on activating transcription factor 5 (ATF5). This is the first in vivo evidence for a role of ATF5 in the mammalian UPR^mt.