The genetic etiology of familial hypobetalipoproteinemia (FHBL) is unclear in the majority of cases. Mutations in apolipoprotein B (APOB) are the only confirmed causes of FHBL. Recently, loss-of-function mutations of PCSK9 gene have been shown to be associated with the hypocholesterolemia phenotype. Our primary goal was to confirm that mutations in PCSK9 could be another cause of FHBL. Using the sequencing approach, we found that the c.43_44insCTG variation in PCSK9, a common in-frame insertion in both African American and Caucasian populations, is associated with the hypocholesterolemia phenotype in three FHBL families. Then we tested whether this variation could be associated with lower cholesterol levels in the general population. A total of 403 subjects from a Caucasian population, in which hypobetalipoprotein (HBL) and normal groups were classified using standard criteria, were sequenced for this variation. The allele frequency of this variation in the HBL group was 0.186, but was only 0.128 in the normal lipid group. The mean plasma low-density lipoprotein (LDL)-cholesterol level in subjects heterozygous for this variant is significantly lower than that in the normal group (po0.01). Heterozygous subjects also had higher high-density lipoprotein (HDL)cholesterol levels (po0.01). In general, LDL-cholesterol concentration in individuals with PCSK9 c.43_44insCTG variation was $10-15 mg/dL lower than that in normal individuals. We conclude that the c.43_44insCTG variant plays a role in lowering cholesterol in the general population. Hum Mutat 27(5),