Lipid antigens are presented to T cells by the non‐polymorphic MHC class I‐related CD1 molecules. Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum (ER)‐resident chaperone that has been shown to lipidate the group 2 CD1 molecule CD1d and thus to regulate its function. We now report that MTP also regulates the function of group 1 CD1 molecules CD1a, CD1b, and CD1c. Pharmacological inhibition of MTP in monocyte‐derived dendritic cells and lymphoblastoid B cell lines transfected with group 1 CD1 resulted in a substantial decrease in endogenous self lipid antigen presentation to several CD1‐restricted T cell lines. Silencing MTP expression in CD1c‐transfected HeLa cells similarly resulted in decreased self reactivity. Unexpectedly, inhibition of ER‐resident MTP, which was confirmed by confocal microscopy, also markedly decreased presentation of exogenous, endosomally loaded, mycobacterial lipid antigens by CD1a and CD1c to T cells. Thus, these studies indicate that MTP, despite its ER localization, regulates endogenous as well as exogenous lipid antigen presentation, and suggest a broad role for MTP in the regulation of CD1 antigen presentation.