Elevated plasma cholesterol is a risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) hinders the uptake of low-density lipoprotein cholesterol (LDL-c) by mediating degradation of LDL receptors (LDLRs) in the liver. Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH). In normolipidemic human plasma, 30-40% of PCSK9 is bound to LDL particles, and this association with LDL inhibits PCSK9’s ability to mediate LDLR degradation in cultured cells. To further investigate the physiological relevance of this interaction, we analyzed natural GOF mutations in PCSK9 and assessed their effects in vitro on LDL binding, LDLR binding and LDLR degradation. Our results indicate that several GOF mutations severely inhibit LDL binding compared to wild type (WT) PCSK9, and only modestly affect LDLR affinity and LDLR degradation. These findings shed light on the potential physiological relevance of the PCSK9-LDL interaction, which may have an inhibitory effect on PCSK9 activity in vivo.