[Aim]  To clarify the role of the HaAChE2 gene in the cotton bollworm (Helicoverpa armigera), and more specifically, to determine if it is the main acetylcholinesterase gene in this pest. [Methods]  We first used qRT-PCR to investigate the expression pattern of the ace1 and ace2 genes. We then constructed an ace2 gene knockout (KO) strain using the CRISPR/Cas9 system and compared the expression of the ace1 gene, AChE activity and the toxicity of six insecticides including phoxim, chlorpyrifos and methomyl, between the knockout strain and the wild-type SCD strain. [Results]  Both ace genes are expressed in different larval stages and tissues. Expression of ace1 was generally higher than that of ace2. A homozygous knockout strain (Haace2-KO) with a ~34 000 bp deletion from exon 1 to exon 5 on the ace2 gene was successfully obtained using CRISPR/Cas9-mediated gene editing and molecular marker-assisted selection. There was no significant difference in the expression of the ace1 gene or AChE activity between the Haace2-KO strain and the wild-type SCD strain (P > 0.05). Bioassay results show that there was no significant difference in the toxicity ratios of phoxim, chlorpyrifos, methomyl, indoxacarb, beta-cyhalothrin and chlorantraniliprole (0.5 to 1.9 fold) between the Haace2-KO strain and the SCD strain. [Conclusion]  The results indicate that knockout of the ace2 gene has no effect on either AChE activity, or anti-cholinesterase insecticide sensitivity, in H. armigera. These findings provide direct evidence that AChE2 is not involved in enzymatic reactions in the nervous system of H. armigera.