Intracellular assembly of chylomicrons (CM) occurs in intestinal enterocytes through a series of complex vesicular interactions. CM are transported from the ER to the Golgi using a specialized vesicular compartment called the prechylomicron transport vesicle (PCTV). In this study, PCTVs were isolated from the enteric ER of the Syrian Golden hamster, and characterized using 2‐DE and MS. Proteomic profiles of PCTV‐associated proteins were developed with the intention of identifying proteins involved in the formation, transport, lipidation, and assembly of CM particles. Positively identified proteins included those involved in lipoprotein assembly, namely microsomal triglyceride transfer protein and apolipoprotein B‐48, as well as proteins involved in vesicular transport, such as Sar1 and vesicle‐associated membrane protein 7. Other groups of proteins found were chaperones, intracellular vesicular trafficking proteins, fatty acid‐binding proteins, and lipid‐related proteins. These findings have increased our understanding of the transport vesicle involved in the intracellular assembly and transport of CM and can provide insight into potential cellular factors responsible for dysregulation of intestinal CM production.