It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of α- and β-subunits of ATP synthase remain unchanged by DAPIT knockdown. These results indicate a critical role of DAPIT in maintaining the ATP synthase population in mitochondria and raise an intriguing possibility of active role of DAPIT in cellular energy metabolism.