PCSK9 is the last member of the proprotein convertases (PCs) family and its gene is mutated in ~ 2% to 3% of individuals with familial hypercholesterolemia (FH). This protein enhances the degradation of the low‐density lipoprotein receptor (LDLR) and hence increases the levels of circulating LDL‐cholesterol (LDLc). Studies of the underlying mechanism(s) regulating the activity of different mutations in the PCSK9 gene are ongoing as they enhance our understanding of the biology and clinical relevance of PCSK9 and its partners. In an attempt to unravel the regulation of PCSK9 transcription and possibly identify mutation ‘hot spot’ regions with alterations in CpG methylation, we present for the first time the complete methylome profile of the PCSK9 gene in modern and archaic humanoids. Our data showed that the genomes of modern humans and archaic PCSK9 exhibit a similar methylation pattern. Next, we defined the mechanistic consequences of three PCSK9 natural mutations (PCSK9‐R96L, ‐R105W, and ‐P174S) and one archaic Denisovan mutation (PCSK9‐H449L) using various complementary cellular and in vitro binding assays. Our results showed that the PCSK9‐H449L is a loss‐of‐function (LOF) mutation, likely due to its lower binding affinity to the LDLR. Similarly, PCSK9‐R96L and ‐R105W are LOF mutations, even though they have been identified in FH patients. The PCSK9‐R105W mutation leads to a significantly lower autocatalytic processing of proPCSK9. PCSK9‐P174S resulted in a LOF in both extracellular and intracellular pathways. In conclusion, our extensive analyses revealed that all studied mutations result in PCSK9 LOF, via various mechanisms, leading to lower levels of LDLc.