7-ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation occur in the lysosomes in a number of cell types, hindering enzymatic transformation, and increasing the chance for lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by transiently transfecting human fibroblasts to overexpress several 7KC-active enzymes. One of our engineered constructs, a lysosomally-targeted cholesterol oxidase that lacked isomerization activity, significantly increased cell viability at concentrations up to 50 µM 7KC relative to controls. Additionally, both overexpression of the lysosomal membrane protein LAMP1 and treatment with 0.9% hydroxypropyl β-cyclodextrin attenuated 7KC-mediated cell death. The overexpression of cholesterol oxidase, sterol 27-hydroxylase, and 11β-hydroxysteroid dehydrogenase, which were localized to the cytoplasm, mitochondria, and endoplasmic reticulum respectively, resulted in either decreased cell viability or no significant change from controls. These results indicate that the lysosome should be the target for treatment of oxysterol-mediated toxicity, and support the utility of using novel lysosomally-targeted enzymes for therapeutic benefit.