A field-collected Aphis gossypii clone [Kushima resistant (KR) clone] was resistant to neonicotinoid insecticides (23.8- to 394-fold). RNA-seq and next-generation sequence analyses were conducted to identify nine cytochrome P450 (CYP) genes that were significantly upregulated in the KR clone as compared with those in the insecticide-susceptible clone. A. gossypii P450s were transiently and efficiently expressed in S2 cell to show that CYP6CY22 (c21228) and CYP6CY13 (c21368), which were the most upregulated of the nine P450s in the KR clone, did not degrade sulfoxaflor, a new class of insecticides acting on insect nAChRs, but markedly metabolized all of the neonicotinoids tested. Hence, P450s are likely to underpin neonicotinoid resistance in other aphids as well in the future, and the P450 expression protocol established here will prompt studies on P450-medidated insecticide resistance and structural analyses of relevant metabolites.