Circulating metabolite levels are biomarkers for cardiovascular disease (CVD). We tested association between rare sequence variants and 226 serum lipoproteins, lipids and amino acids in 7,142 healthy participants. Gene-based association analyses identified novel gene-trait associations with ACSL1 , MYCN , FBXO36 and B4GALNT3 ( p <2.5 × 10 −6 ), and confirmed established associations. Regulation of the pyruvate dehydrogenase (PDH) complex was associated for the first time, in gene set analyses, with IDL and LDL parameters, as well as circulating cholesterol ( p METASKAT <2.41 × 10 −6 ). Individuals at the lower tails of the distributions of four out of 49 lipoproteins and lipids had an excess of predicted deleterious variants in lipoprotein disorder and metabolism gene sets ( p permutation <0.00037). These four traits were CVD risk factors (e.g. S-VLDL-C), demonstrating that rare “protective” variation is a significant contributor to lipoprotein levels in a healthy population. In conclusion, rare variant analysis of these important metabolic biomarkers reveals novel loci and pathways involved in their regulation.