Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E-deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.