The sex pheromone biosynthetic pathways of lepidopterans require the concerted actions of multiple gene products. A number of pheromone gland (PG)-specific genes have been cloned in recent years and, whereas in vitro characterizations have indicated functions consistent with roles in pheromone production, there have been no clear demonstrations in vivo. Using an RNA interference-mediated loss-of-function approach, we injected newly formed Bombyx mori pupae with dsRNAs corresponding to genes of interest [i.e., PG fatty acyl reductase (pgFAR), B. mori PG Z11/Delta10,12 desaturase (Bmpgdesat1), PG acyl-CoA-binding protein (pgACBP), midgut ACBP, and pheromone biosynthesis activating neuropeptide receptor (PBANR)] to assess their specific roles during pheromonogenesis. In all cases, the introduced dsRNAs induced a dose-dependent reduction in sex pheromone production with the corresponding decrease in transcript levels. No effects on pupal development or adult emergence were observed. Disrupting the PBANR gene resulted in a loss of the lipase activity that liberates pheromone precursors, whereas knockout of the pgACBP gene prevented the daily accumulation and fluctuation of the triacylglycerols that function as the cellular deposits for the pheromone precursors. Taken together, our results provide unequivocal evidence that the pgACBP, Bmpgdesat1, pgFAR, and PBANR gene products are essential during pheromonogenesis and demonstrate the power of this methodology for dissecting the molecular interactions that comprise biosynthetic pathways.