Hepatocellular carcinoma (HCC) is a primary liver cancer with high mortality. Paeoniflorin is a pinane monoterpene picroside with anti-tumor effect isolated from Chinese peony root and white peony root. To investigate the underlying mechanism of paeoniflorin (PF) regulating hepatocellular carcinoma (HCC) progression via 5-hydroxytryptamine receptor 1D (5-HT1D). HepG2 and SMMC-7721 hepatoma cells were treated with different concentrations of PF (0, 5, 10, 20 μM). Cell proliferation, apoptosis, migration, and invasion were examined by CCK-8 and colony formation assays, flow cytometry, wound healing assay, and transwell assay, respectively. RT-qPCR assay was used to detect the expression level of 5-HT1D, and Western blot assay was used to detect the expressions of 5-HT1D and Wnt/β-catenin pathway-related proteins. With the increase of PF concentration, the mRNA level of 5-HT1D in HepG2 and SMMC-7721 hepatoma cells were decreased in a dose-dependent manner, and the proliferation, colony formation, migration and invasion ability of cells were gradually weakened, while the apoptosis rate was gradually increased. Overexpression of 5-HT1D significantly promoted the proliferation, colony formation, migration and invasion of HepG2 and SMMC-7721 cells, and increased the expression of Wnt/β-catenin pathway-related proteins β-actenin, survivin, C-myc, and Cyclin D1. Furthermore, 5-HT1D overexpression could reverse the effect of PF on hepatoma cells and inhibit the expressions of Wnt/β-catenin pathway-related proteins. PF may inhibit the progression of HCC by blocking Wnt/β-catenin pathway expression through downregulating 5-HT1D. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.