Updated project metadata. Digested dietary fats are taken up, processed and transported by enterocytes to supply the body with lipids. Most absorbed lipids are assembled into pre-chylomicrons in the endoplasmic reticulum (ER) of enterocytes, which are then transported to the Golgi for maturation and subsequent secretion to the circulation. The role of mitochondria in regulating intestinal lipid transport remains unknown. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with intestinal epithelial cell (IEC)-specific ablation of the mitochondrial-specific aspartyl - tRNA synthetase DARS2, as well as of the respiratory chain subunit SDHA or the assembly factor COX10 failed to thrive and showed massive accumulation of lipids within large lipid droplets (LDs) in enterocytes of the proximal small intestine (SI). Feeding a fat-free diet inhibited the formation of LDs in DARS2-deficient enterocytes, showing that accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in IECs. Moreover, DARS2-deficient enterocytes showed a distinct lack of mature chylomicrons concomitant with a disorganisation of the Golgi apparatus, suggesting that impaired ER to Golgi trafficking underlies impaired chylomicron production and secretion. Taken together, these results revealed a vital role of mitochondria in regulating dietary lipid transport in enterocytes, which is relevant for understanding the intestinal and nutritional defects observed in patients with mitochondrial defects.