Cytochrome P450 monooxygenase (CYP), an important detoxifying enzyme in insects, is involved in the metabolism and degradation of both exogenous compounds and endogenous substances. To investigate the involvement of the Tribolium castaneum TcCYP4C1 gene in detoxification metabolism under high CO₂ and the protective role of trehalose against hypoxic stress in insects. In the present study, after successfully silencing the TcCYP4C1 gene of T. castaneum by double-stranded RNA(dsRNA), the larvae were exposed to 95% CO₂. This exposure resulted in a statistically significant increase in larval mortality and a significant elevation in the activity of the carboxylesterase enzyme (CarE). However, a decrease in mortality from 18.15% to 11.24% was observed when larvae were fed trehalose after dsRNA injection. In addition, the gene expression levels of the trehalose metabolism pathway-related genes TRE1-3, TRE1-4, and TPS2 were significantly up-regulated after 95% CO₂ treatment. In summary, the TcCYP4C1 gene emerges as a pivotal factor in the adaptive response of T. castaneum to high CO₂. Trehalose effectively mitigates the detrimental effects resulting from the silencing of TcCYP4C1 and exposure to high CO₂ stress in T. castaneum. Our findings not only establish a theoretical foundation for the development of novel pesticides tailored for low-oxygen grain storage environments but also inspire innovative, environmentally sustainable pest management strategies in the grain storage sector.