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Acetyl-CoA, the precursor of sex pheromone biosynthesis in Helicoverpa armigera, is generated from glycolysis. As the first speed-limited enzyme in glycolysis, Hexokinase (HK) plays an important role in acetyl-CoA production. However, the function of HK in sex pheromone production remains unclear. This study employed H. armigera as material to explore the role of HK in sex pheromone production. Results demonstrated that the transcription profile of HaHK in female moth pheromone glands (PGs) was consistent with the release fluctuation of sex pheromone. Interference of HaHK prevented the increase of acetyl-CoA content induced by PBAN. Therefore, knockdown of HaHK in female PGs caused significant decreases in (Z)-11-hexadecenal (Z11-16:Ald) production, female capability to attract males, and mating rate. Furthermore, sugar feeding (5% sugar) increased the transcription and enzymatic activity of HK. Pheromone biosynthesis activating neuropeptide (PBAN) signal phospho-activated HaHK in PGs and Sf9 cells via protein kinase A (PKA), as shown by pharmacological inhibitor analysis. In general, our study confirmed that PBAN/cAMP/PKA signal activated HaHK, in turn promoted glycolysis to ensure the supply of acetyl-CoA, and finally facilitated sex pheromone biosynthesis and subsequent mating behavior.