Single nucleotide polymorphisms (SNPs) that alter exon splicing efficiency are an emerging class of functional genetic variants. Since mutations in low-density lipoprotein receptor (LDLR) are a primary cause of familial hypercholesterolemia, we evaluated whether LDLR SNPs may alter splicing efficiency and cholesterol homeostasis. A SNP within LDLR exon 12, rs688, was identified in silico as neutralizing a putative exon splicing enhancer. Studies in human liver samples established that this SNP was associated with significantly decreased LDLR exon 12 splicing efficiency in women in vivo. In vitro minigene splicing studies qualitatively replicated these in vivo results and demonstrated that rs688 specifically modulates splicing efficiency. These effects on splicing may be physiologically relevant because the presence of the rs688 minor allele associates with increased total and LDL-cholesterol in female members of the Framingham Offspring Study. The largest rs688-associated cholesterol differences were observed in pre-menopausal women. In summary, these studies identify an LDLR SNP present in approximately 60% of Caucasians that is associated with significant 10% increases in total and LDL-cholesterol in pre-menopausal women.