Coenzyme Q10 (CoQ10) is a key component of the mitochondrial electron transfer chain and is one of the most important antioxidants. We previously found that glycoprotein prosaposin (Psap) binds CoQ10 in human cells. Although Psap is expressed in the intestines, its role in the gastrointestinal tract is not clear. To elucidate the role of Psap in the intestines, we established Psap knockdown (KD) Caco-2 cells, which are an intestinal epithelial cell line. Cellular CoQ10 levels decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Cellular ATP levels also decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Lower ATP levels in the intestines have been reported to result in the failure of tight junction formation. As expected, Psap KD Caco-2 monolayers did not produce transepithelial electrical resistance, while parental Caco-2 monolayers did. Moreover, a fluorescent dye, lucifer yellow, leaked out through Psap KD Caco-2 monolayers, whereas it did not through parental Caco-2 monolayers. These results indicate that Psap is essential to maintain cellular levels of CoQ10 and ATP, and consequently to form tight junctions in the gastrointestinal tract.