Doxorubicin (DOX) is an effective anti-cancer agent. However, DOX treatment increases patient susceptibility to dilated cardiomyopathy. DOX predisposes cardiomyocytes to insult by suppressing mitochondrial energy metabolism, altering calcium flux, and disrupting proteolysis and proteostasis. Prior studies have assessed the role of macroautophagy in DOX cardiotoxicity; however, limited studies have examined whether DOX mediates cardiac injury through dysfunctions in inter- and/or intra-lysosomal signaling events. Lysosomal signaling and function is governed by transcription factor EB (TFEB). In this study, we hypothesized whether DOX caused myocyte injury by impairing lysosomal function and signaling through negative regulation of TFEB. Indeed, we found that DOX consistently repressed cellular TFEB expression, which was associated with impaired cathepsin proteolytic activity across in vivo , ex vivo , and in vitro models of DOX cardiotoxicity. Furthermore, we observed that loss of TFEB was associated with reduction in macroautophagy protein expression, inhibition of autophagic flux, impairments in lysosomal cathepsin B activity, and activation of cell death. Restoration and/or activation of TFEB in DOX-treated cardiomyocytes prevented DOX-induced suppression of cathepsin B activity, reduced DOX-mediated ROS overproduction, attenuated activation of caspase-3 and improved cellular viability. Loss of TFEB inhibits lysosomal autophagy, rendering cardiomyocyte susceptible to DOX-induced proteotoxicity and injury. Collectively, our data reports a novel mechanism wherein DOX primes cardiomyocytes for cell death by depleting cellular TFEB.