Leonurus japonicus is a vital herb known for its blood-activating and menstruation-regulating properties, with stachydrine being a crucial secondary metabolite contributing to these effects. Enhancing the accumulation of active ingredients is a key concern in cultivation. This study utilized a hydroponic experiment with varying inorganic nitrogen (N) concentrations (supplied as Ca(NO₃)₂·4H₂O and KNO₃) as nutrient substrates, demonstrating that exogenous nitrogen input significantly increased biomass and promoted stachyridine synthesis in L. japonicus over an extended incubation period. Analysis of key intermediates in the stachydrine synthesis pathway suggests that exogenous nitrogen enhanced the amino acid synthesis pathway in primary metabolism, promoting glycolysis and accumulating tricarboxylic acid (TCA) cycle substances, namely, pyruvate and α-ketoglutarate. This, in turn, enhanced the production of stachydrine precursors—glutamic acid and ornithine. Nitrogen’s involvement redirected the amino acid synthesis pathway more towards the alkaloid synthesis pathway. The study confirmed the essential role of nitrogen in promoting the quality formation of L. japonicus and provided initial evidence for the mechanism behind stachydrine formation, offering theoretical and practical insights for improving L. japonicus cultivation technology.