Background— A low level of plasma high-density lipoprotein cholesterol (HDL-C) is a risk factor for cardiovascular disease. HDL particles are modulated by a variety of lipases, including endothelial lipase, a phospholipase present on vascular endothelial cells. The proprotein convertase subtilisin/kexin type 5 ( PCSK5 ) gene product is known to directly inactivate endothelial lipase and indirectly cleave and activate angiopoetin-like protein 3, a natural inhibitor of endothelial lipase. We therefore investigated the effect of human PCSK5 genetic variants on plasma HDL-C levels. Methods and Results— Haplotypes at the PCSK5 locus were examined in 9 multigenerational families that included 60 individuals with HDL-C PCSK5 gene in 12 probands with HDL-C P P =0.002 and P =0.005, respectively). The SNP rs11144782 was also associated with very low-density lipoprotein ( P =0.039), triglycerides ( P =0.049), and total apolipoprotein levels ( P =0.022). In stage 2, we replicated the association of rs11144766 with HDL-C ( P =0.014) in an independent sample of Finnish low HDL-C families. In a combined analysis of both stages (n=883), region-wide significance of rs11144766 and low HDL-C was observed (unadjusted P =1.86�10 −4 and Bonferroni-adjusted P =0.031). Conclusions— We conclude that variability at the PCSK5 locus influences HDL-C levels, possibly through the inactivation of endothelial lipase activity, and, consequently, atherosclerotic cardiovascular disease risk.