Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit flyDrosophila melanogaster, both C27and C28ecdysteroids have been identified. While the biosynthetic pathway of the C27ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C28ecdysteroids from C28or C29dietary sterols remains unknown. In this study, we found that different dietary sterols (including the C27sterols cholesterol and 7-dehydrocholesterol, the C28sterols brassicasterol, campesterol, and ergosterol, and the C29sterols β-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression inD. melanogasterKc cells. We also found that a single dietary sterol was sufficient to supportD. melanogastergrowth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, our study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C27ecdysteroid 20E biosynthetic pathway also participated in the conversion of C28and C29dietary sterols into C28ecdysteroids.