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The wheat aphid Sitobion miscanthi is a dominant and destructive pest in agricultural production. Insecticides are the main substances used for effective control of wheat aphids. However, their extensive application has caused severe resistance of wheat aphids to some insecticides; therefore, exploring resistance mechanisms is essential for wheat aphid management. In the present study, CYP6CY2, a new P450 gene, was isolated and overexpressed in the imidacloprid-resistant strain (SM-R) compared to the imidacloprid-susceptible strain (SM-S). The increased sensitivity of S. miscanthi to imidacloprid after knockdown of CYP6CY2 indicates that it could be associated with imidacloprid resistance. Subsequently, the posttranscriptional regulation of CYP6CY2 in the 3′ UTR by miR-3037 was confirmed, and CYP6CY2 participated in imidacloprid resistance. This finding is critical for determining the role of P450 in relation to the resistance of S. miscanthi to imidacloprid. It is of great significance to understand this regulatory mechanism of P450 expression in the resistance of S. miscanthi to neonicotinoids. [Display omitted] • CYP6CY2, a new P450 gene was isolated. • CYP6CY2 was overexpressed in the imidacloprid-resistant strain (SM-R) compared to that in the imidacloprid-susceptible strain (SM-S). • The increased sensitivity to imidacloprid after knockdown of CYP6CY2. • CYP6CY2 in the 3′ UTR regulated by miR-3037 posttranscriptionally. • CYP6CY2 regulated by miR-3037 involved in imidacloprid resistance. [ABSTRACT FROM AUTHOR]