Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II enzymes that conjugate a variety of small lipophilic molecules with UDP sugars and alter them into more water-soluble metabolites. Therefore, glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of thiamethoxam against the resistant strain of Aphis gossypii, which indicates that UGTs are involved in thiamethoxam resistance in the cotton aphid. Based on transcriptome data, 31 A. gossypii UGTs belonging to 11 families (UGT329, UGT330, UGT341, UGT342, UGT343, UGT344, UGT345, UGT348, UGT349, UGT350, and UGT351) were identified. Compared with the thiamethoxam-susceptible strain, the transcripts of 23 UGTs were elevated, and the transcripts of 13 UGTs (UGT344J2, UGT348A2, UGT344D4, UGT341A4, UGT343B2, UGT342B2, UGT350C3, UGT344N2, UGT344A14, UGT344B4, UGT351A4, UGT344A11, and UGT349A2) were increased by approximately 2.0-fold in the resistant cotton aphid. The suppression of selected UGTs significantly increased the insensitivity of resistant aphids to thiamethoxam, suggesting that the up-regulated UGTs might be associated with thiamethoxam tolerance. This study provides an overall view of the possible metabolic factor UGTs that are relevant to the development of insecticide resistance. The results might facilitate further work to validate the roles of these UGTs in thiamethoxam resistance.