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In view of the prevailing insecticide resistance and increasing safety and health concerns, there is an urgent need to develop effective and environmentally friendly methods to control stored-product pests. S-methoprene, a microbial volatile compound with insecticidal properties, has been identified as a potential candidate for this purpose. However, its efficacy against storage pests is not well understood. In this study, wheat was treated with S-methoprene at a rate of 5 mg/kg and stored in ambient conditions within a grain bin. Samples were collected after 90 and 365 days, and bioassays were conducted on 20 mixed-sex developmental stages of Tribolium castaneum and T. confusum. S-methoprene showed significant ovicidal, pupicidal, and adulticidal activity against T. castaneum and T. confusum. The efficacy of S-methoprene was found to be influenced by the developmental stage of the pests and the timing of its application. In the 90-day treatment group, the hatching rate of three T. castaneum strains was zero, while that of T. confusum strains from Guangdong and Beijing was (1.67±2.04)% and 0%, respectively. S-methoprene treatment in the 90-day group resulted in effective pupicidal activity against both T. castaneum and T. confusum, with the F1 generation of adults being unable to reproduce. However, in the 365-day treatment group, the pupicidal activity significantly decreased, and the F1 generation of adults were able to reproduce but their F1 generation pupae can not hatch. The adulticidal activity of both pests had some effects in both treatments, but the mortality rate significantly decreased in the 365-day treatment group. The F1 generation of T. castaneum only consisted of larvae, while some larvae of T. confusum were able to pupate but could not further develop into adults. T. castaneum was found to be more susceptible to S-methoprene than T. confusum. After S-methoprene induction, the activity of three detoxification enzymes in Tribolium species was affected in varying degrees and had different variation patterns. The activity was positively correlated with induction time.