Arthropods, including insects, convert sterols into cholesterol due to the inability to synthesise cholesterol de novo. 24-dehydrocholesterol reductase (DHCR24) plays an important role in the conversion. Not only involving the cholesterol biosynthesis in vertebrates, DHCR24 is required for the conversion of desmosterol into cholesterol in phytophagous insects. The current study extensively examined DHCR24 in omnivorous insects, which feed on both plants and animals, using Gryllus bimaculatus as the experimental model. We identified cDNAs encoding two homologues of DHCR24 from G. bimaculatus, which were designated as GbDHCR24-1 and GbDHCR24-2. Both homologues contained the flavin adenine dinucleotide binding domain, which is a feature of DHCR24. Quantitative polymerase chain reaction revealed that among tissues of adult crickets, fat body and anterior midgut expressed high levels of GbDHCR24s. Both fat body and anterior midgut demonstrated DHCR24 activities in which one of the functions is the conversion of desmosterol into cholesterol in vitro. Knockdown of GbDHCR24-1 significantly reduced the conversion activity in the anterior midgut while knockdown of the GbDHCR24-2 did not. Additionally, the accumulation of desmosterol was detected in a feeding experiment with a specific DHCR24 inhibitor, azacosterol. We finally concluded that GbDHCR24-1 is the major enzyme that facilitates the desmosterol-to-cholesterol-conversion in crickets.