Plant-parasitic nematodes ingest and convert host phytosterols via dealkylation to cholesterol for both structural and hormonal requirements. The insect 24-dehydrocholesterol reductase (DHCR24) was shown in vitro as a committed enzyme in the dealkylation via chemical blocking. However, an increased brood size and ovulation rate, instead compromised development, were observed in the engineered nematode Caenorhabditis elegans where the DHCR24 gene was knocked down, indicating the relationship between DHCR24 and dealkylation and their function in nematodes remains illusive. In this study, a defect in C. elegans DHCR24 causes impaired growth of the nematode with sitosterol (a major component of phytosterols) as a sole sterol source. Plant sterols with rationally designed structure (null substrates for dealkylation) can't be converted to cholesterol in wild-type worms, and their development was completely halted. This study underpins the essential function of DHCR24 in nematodes and would be beneficial for the development of novel nematocidal strategies.