Abstract Background：Pseudostellaria heterophyllais one of the most well-known traditional Chinese medicines, and its secondary metabolites play an important medicinal role. Paclobutrazol (PBZ), an antagonist of the plant hormone gibberellin, is widely used to increase the yield of P. heterophylla. Whether PBZ affects the synthesis and accumulation of the plant’s secondary metabolites is unclear. Here we investigated the effects of PBZ on the production of polysaccharides, saponins and heterophyllin B in the root tubers of P. heterophylla, and further explored its molecular mechanisms. Results：We found that PBZ promoted the yield of P. heterophylla, but reduced the accumulation of polysaccharides, saponins and heterophyllin B. PBZ dramatically downregulated genes associated with gibberellin synthesis and reduced concentration of the hormone in root tubers. Inhibition of gibberellin signaling by PBZ altered the expression of genes involved in synthesis and metabolism of polysaccharides, saponins and heterophyllin B.Exogenous gibberellin reversed these effects. Conclusion：These results suggest that PBZ suppresses the synthesis of saponins, polysaccharides and heterophyllin B by antagonizing gibberellin signaling in P. heterophylla, providing important guidance for its cultivation.