This review provides the latest understanding in different aspects of VLDL assembly process, including structure-function relationships within apoB, mutations in APOB causing hypobetalipoproteinemia, significance of modulating microsomal triglyceride-transfer protein activity in VLDL assembly, alterations of VLDL assembly by different fatty acid species, and hepatic proteins involved in vesicular trafficking, and cytosolic lipid droplet metabolism that contribute to VLDL assembly. The gene discussed is APOB; the disease is hypobetalipoproteinemia.