The importance of glutathione S-transferases (GSTs) in imidacloprid resistance in Nilaparvata lugens, a major rice pest, and other insects was often excluded, mostly due to the slight effects of diethyl maleate (DEM) on synergizing imidacloprid in resistant populations. Here, we found that the synergistic effects of DEM were time-dependent. At 24 or 48 h, the time often selected to record mortalities in imidacloprid bioassay, DEM really did not cause an obvious increase in imidacloprid toxicity. However, significant effects were observed after 72 h. The results revealed that GSTs, as phase II detoxification enzymes to metabolize secondary products generated from phase I detoxification enzymes, were also important in imidacloprid resistance in N. lugens, but might have occurred a little later than that of P450s and CarEs as phase I enzymes. The constitutive overexpression in the imidacloprid-resistant strain G25 and expression induction by imidacloprid in the susceptible strain S25 indicated that four GST genes, NlGSTs1, NlGSTs2, NlGSTe1, and NlGSTm1, were important in imidacloprid resistance, which was confirmed by RNAi test. The higher expression levels and more expression induction by imidacloprid in the midgut and fat body compared to the whole insect supported the important roles of these four GSTs, which was also supported by the more overexpression times in the midgut and fat body versus the whole insect between G25 and S25 strains. Taking the data together, the study ascertained the roles of GSTs in imidacloprid resistance in N. lugens.