Abstract: For effectively control of Mythimna separate Walker by reasonable application of emamectin benzoate, the cross-resistance of emamectin benzoate resistant population to other five insecticides and its biochemical resistance mechanisms were studied by bioassay and biochemical assay. The results revealed that medium level of cross-resistance to abamectin (resistance ratio:RR₅₀=21.80), chlopyrifos (RR₅₀=17.68) and methomyl (RR₅₀=10.85), and low level of cross-resistance to phoxim (RR₅₀=6.00) and cyfluthrin (RR₅₀=5.65) for emamectin benzoate resistant population. The enzyme inhibitors piperonyl butoxide (PBO), diethyl maleate (DEM), S, S, S-tributyl phosphorotrithioate (DEF) and triphenyl phosphate (TPP) had significant synergism to emamectin benzoate in both resistant and susceptible populations. The contents of cytochrome P450 and b₅, and activities of O-demethylase, glutathione S-transferases and carboxylesterase of the emamectin benzoate resistant population were 3.23, 3.65, 3.63, 1.64 and 2.66-fold of that in the susceptible population, respectively. These results indicated that the increased detoxification enzyme activities may play an important role in the resistance of M. separate to emamectin benzoate.