The microsomal triglyceride transfer protein (MTP), along with its partner, protein disulphide isomerase, performs a wide range of lipid transport functions necessary for maintenance of whole-body lipid homeostasis. In this review, we summarize the recent deluge of comparative and functional genomic data that have forced a radical re-appraisal of the evolutionary processes that established the major lipid transport pathway in man, and the different structural and lipid transfer roles MTP plays within it. This is followed by an overview of MTP structure-function relationships, highlighting two newly identified functional roles: first, the production of small, apolipoprotein (apo)B-containing lipoprotein particles in cardiac myocytes and, second, the lipidation of a major histocompatibility complex class-I related molecule (CD1d) that presents glycolipid antigens to distinct subsets of natural killer T cells. We also discuss the interactions of MTP with proteins such as apoB and CD1d, and the complex mechanisms regulating MTP transcription in different cell types and nutritional states. The past five years has witnessed remarkable progress in teasing out the different functionalities of MTP, and the properties of the different molecules that inhibit MTP activity, data that are likely to underpin the design of the next generation of MTP/apoB inhibitors for preventing cardiovascular disease attributable to the increased production of atherogenic lipoproteins.