Abstract  [Aim]  To investigate the effects of heat acclimation (27 ℃) on insecticide susceptibility and detoxification enzyme activity of Rhopalosiphum padi. [Methods]  The leaf-dipping method, enzyme activity assay and real-time quantitative PCR (RT-qPCR) were used to measure insecticide susceptibility, detoxification enzymes activity, and gene expression, respectively, in R. padi. [Results]  Heat acclimation had a significant effect on the susceptibility of R. padi to six insecticides, with acetamiprid having the highest index of relative toxicity (4.59). Synergist experiment showed that diethyl maleate (DEM) had no significant synergistic effect on acetamiprid in either the control (CK) or heat acclimation (HA) strains. Conversely, piperonyl buoxide (PBO) had a significant synergistic effect on acetamiprid in both CK and HA strain, with synergism ratios of 3.95 and 22.41, respectively. Moreover, triphenyl phosphate (TPP) had a significant synergistic effect on acetamiprid in the HA strain, with a synergism ratio of 4.82. The activity of cytochrome P450 (P450) in the HA strain was significantly higher than CK strain. RT-qPCR revealed that the expression of CarE (2.1, P=0.037), GSTO (1.7, P=0.035), CYP6CY7 (7.0, P=0.003) and CYP6CZ1 (1.9, P=0.013) was significantly upregulated in the HA strain, while CYP6CJ1 (0.40, P=0.026) was significantly downregulated in the HA strain. [Conclusion]  Heat acclimation significantly increased the sensitivity of R. padi to acetamiprid, and the synergism of piperonyl butoxide and triphenyl phosphate on acetamiprid. Heat acclimation significantly increased the activity of detoxifying enzyme P450 and induced the differential expression of five detoxification enzyme genes.