Background and Aims Cholangiocarcinoma, a rare malignant tumor, is difficult to diagnose and often detected at an advanced stage, limiting treatment options to palliative care. Conventional chemotherapy drugs have poor efficacy against cholangiocarcinoma, making the search for new treatments critical. This study was conducted to investigate the effects of Huai'erjunzhi on the malignant biological behavior of human cholangiocarcinoma cells and its relationship with the TGF-β/Smad pathway, aiming to provide a theoretical basis for the use of Huai'erjunzhi in cholangiocarcinoma treatment.Methods Human normal liver cells (L-02) and human cholangiocarcinoma cells (HuCCT1) were incubated with different concentrations of Huai'erjunzhi for various durations. Cell proliferation was assessed, and the half-maximal inhibitory concentration (IC₅₀) was calculated. HuCCT1 cells were divided into a negative control group (no intervention), a positive control group (15 mg/L cisplatin), and different Huai'erjunzhi intervention groups (1/5 IC₅₀, 2/5 IC₅₀, and IC₅₀ based on preliminary experimental results). Scratch and Transwell assays were used to measure cell migration and invasion, while Western blot was employed to detect the expression of proteins related to the TGF-β/Smad pathway in HuCCT1 cells.Results Only high concentrations of Huai'erjunzhi (>312.5 mg/L) significantly inhibited the proliferation of L-02 cells. Huai'erjunzhi significantly inhibited the proliferation of HuCCT1 cells at concentrations above 5 mg/L in a concentration-dependent manner (all P<0.05), with IC₅₀ values of 138.52 mg/L at 24 h, 99.41 mg/L at 48 h, and 113.52 mg/L at 72 h. Compared with the negative control group, the positive control group and the three Huai'erjunzhi intervention groups (20, 40, and 100 mg/L) exhibited reduced migration distance, decreased invasive cell numbers, lower expression of TGF-β1, Smad2, Smad3, Smad4, N-cadherin, Snail, and Slug, and increased expression of E-cadherin (all P<0.05). Compared with the positive control group, these changes in the Huai'erjunzhi groups were less pronounced but showed a clear concentration-dependent relationship (all P<0.05).Conclusion Huai'erjunzhi can potentially inhibit the malignant biological behavior of HuCCT1 cells by inhibiting the TGF-β/Smad pathway.