Evolutionary adaptations of herbivorous insects are often dictated by the necessity to withstand a corresponding evolutionary innovation in host plant defense. Glucosinolate sulfatase (GSS) enzyme activity is considered a central adaptation strategy in Plutella xylostella against glucosinolates (GS)-myrosinase defense system in the Brassicales. The high functional versatility of sulfatases suggests that they may perform other vital roles in the process of growth and development. Here, we used a CRISPR/Cas9 system to generate stable homozygous single/double mutant lines of gss1 or/and gss2 with no predicted off-target effects, to analyze the functions of the pair of duplicated genes in the development and host adaptation of P. xylostella. The bioassays showed that, when fed on their usual artificial diet, significant reduction in egg hatching rate and final larval survival rate of the single mutant line of gss2 compared with the original strain or mutant lines of gss1, revealing unexpected functions of GSS2 in embryonic and larval development. When larvae of homozygous mutant lines were transferred onto a new food, Arabidopsis thaliana, no induced effect at protein level of GSS1/2 or gene expression level of gss1/gss2 was detected. The absence of GSS1 or GSS2 reduced the survival rate of larvae and prolonged the duration of the larval stage, indicating that both GSS1 and GSS2 played an important role in adaptation to host plants. The versatile functions of duplicated GSSs in this study provide a foundation for further research to understand potential functions of other sulfatase members and support evidence of adaptation in herbivorous insects.,Copyright © 2020. Published by Elsevier Ltd.