The daily susceptibility rhythm of the cotton aphid, Aphis gossypii Glover, to diazinon and the corresponding changes in the activity of three xenobiotic detoxifying enzymes—cytochrome P450 monooxygenases (P450), glutathione S-transferases (GSTs), and esterases—were investigated. Bioassays were conducted to estimate the median lethal doses (LD₅₀) of diazinon at six different zeitgeber times (ZT0, 4, 8, 12, 16, and 20) under constant light (LL) and lighting conditions of 16 h of light and 8 h of darkness (LD). The results showed that the highest susceptibility occurred at the onset of night (ZT16) and 4 h before this time point (ZT12) under the LD condition. The endogenous rhythmicity of susceptibility was ensured, as the highest susceptibility occurred at the same time under the LL condition. The circadian changes in susceptibility to diazinon were almost coincident with changes in esterase and GSTs activity, but not in P450 activity. We also found rhythmic changes in energy components of whole-body aphids, with similar patterns of circadian changes of proteins, lipids, and soluble carbohydrates, but not glycogen, under LL and LD conditions. These photoperiod conditions (LD and LL) showed different fluctuation in trends of energy resources and of course, different quantities. Our study represents the first report of circadian control of insecticide susceptibility in aphids and provides insights into more efficient control of these pests by unveiling the times of day during which aphids are more susceptible to insecticides with attention to endogenous physiological phenomena.