Phytoseiulus persimilis is a specialist predator of spider mites, with promising control effects. Its population generally has a stable sex ratio. Under appropriate conditions, the proportion of female offspring can reach 80%. But the mechanisms of sex determination in P. persimilis is still unclear. Sex-lethal (sxl) is an important gene in studying sex regulation mechanisms in insects. In the model organism Drosophila melanogaster, it has been proved to be a switch controlling sex determination. Herein, we screened one sex-lethal gene ortholog in the genome of P. persimilis (Ppsxl). We separated male and female offspring successfully based on the order of the egg laid, and found Ppsxl is expressed in both sexes at different stages. The expression peak was found in females 10 h after mating, and it was also highly expressed in eggs that developed into males. RNA interference was used to study the function of Ppsxl. When Ppsxl was interfered, all female individuals remained fertile. However, their cumulative fecundity decreased by 18.0%, daily fecundity decreased by 13.4%, and the proportion of female offspring decreased by 22.7%. This study showed that Ppsxl gene has a certain influence on the reproduction of P. persimilis, but its influence on sex determination may not be as critical as that in D. melanogaster. The regulation of sxl on P. persimilis was different from that in D. melanogaster.