Pheromones are environmentally friendly alternatives to traditional pesticides for pest control. They are widely applied for insect monitoring, mating disruption and mass trapping. Nicotiana benthamiana and N. tabacum are potential green biomass production platforms of moth sex pheromones. Using these two Nicotiana species as plant factories, we expressed biosynthetic genes of plant and insect origin in leaf tissue. Moth sex pheromone precursors (E)-11-tetradecenoic acid, (Z)-11-tetradecenoic acid and (Z)-11-hexadecenoic acid were produced by introducing the acyl-ACP thioesterases CpuFatB1 from Cuphea pulcherrima or CpaFatB2 from C. palustris and the fatty acyl desaturases Ave∆11 from Argyrotaenia velutinana, CpaE11 from Choristoneura parallela or Atr∆11 from Amyelois transitella, under the control of CaMV-35S promoter. Among the Nicotiana spp. transformants, the best line produced (Z)-11-hexadecenoic acid at 17.6% of total fatty acids in leaves, during flowering stage, corresponding to 335 µg of (Z)-11-hexadecenoic acid per gram of fresh leaf. The (Z)-11-hexadecenoic acid production lines from N. benthamiana were selected for further propagation to obtain homozygous lines. In the N. benthamiana T2 generation, the production quantity of (Z)-11-hexadecenoic acid was stable. Our study demonstrates the feasibility of stable transformation of N. benthamiana for production of moth pheromone precursors in vegetative tissue.