10-Hydroxy-2-decenoic acid (10-HDA), a major fatty acid (FA) component of royal jelly, is synthesized in the mandibular glands (MGs) of worker honeybees. Despite its well-documented nutritional and therapeutic significance, the biosynthetic pathway and regulatory mechanisms of 10-HDA production remain largely unresolved. In this study, the molecular basis of 10-HDA biosynthesis and regulation in the MGs of newly emerged bees (NEBs), nurse bees (NBs), and forager bees (FBs) were investigated using RNA sequencing and weighted gene co-expression network analysis (WGCNA). A five-step biosynthetic pathway for 10-HDA was proposed, and cross-species analysis of Apis mellifera and A. cerana revealed the conserved expression patterns of 15 key enzymes involved. Functional validation via RNA interference (RNAi) demonstrated that knockdown of acyl-CoA Delta(11) desaturase (d11ds, LOC551527), a key enzyme in FA desaturation, led to a 50% reduction in 10-HDA levels. Protein–protein interaction (PPI) network analysis further identified transcriptional regulators Kay and Drep-2 as potential modulators of 10-HDA metabolism. This study provides the first comprehensive mechanistic model of 10-HDA biosynthesis in honeybee MGs and highlights the labor-specific regulation of FA metabolism. These findings offer promising genetic targets for improving the royal jelly quality through genetic technology.