Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme with a typical function of degrading the neurotransmitter acetylcholine. Although two ace genes were reported in Plutella xylostella, their function differences remain largely unknown. The chemically synthesized siRNAs (si‐Pxace1 and si‐Pxace2) were injected into the second instar larvae to knock down Pxace1 and Pxace2, either respectively or simultaneously. The mRNA abundance of Pxace1 and Pxace2 was significantly reduced to 7–33.5% of the control levels at 72 h after siRNA injection. The AChE activities were significantly decreased at 96 h after treatment. Silencing of Pxace1 or Pxace2 resulted in mortality of 33.9 and 22.9%, respectively. The survivors in siRNA‐treated groups had apparent growth inhibition such as reduction in larvae weights and lengths, malformation and motor retardation. Knockdown of Pxace1 apparently affected more on larvae growth than that of Pxace2, suggesting that Pxace1 had more important roles than Pxace2. Both Pxace1 and Pxace2 genes might have atypical functions in regulating larvae growth and motor ability.