This study aimed to investigate the potential anticancer mechanisms of notopterol in triple-negative breast cancer (TNBC) through modulation of the indoleamine 2,3-dioxygenase (IDO)-mediated tryptophan metabolic pathway via Janus kinase 2 (JAK2). Intracellular kynurenine levels were measured to evaluate the effects of different concentrations of notopterol on kynurenine production. Cell viability was assessed using the CCK-8 assay. Cells were divided into the control group, interferon-γ (IFN-γ) group, JAK inhibitor I group, and notopterol groups (5, 10 and 20 μmol/L). RT-qPCR was performed to determine IDO1 mRNA expression, while western blot analysis was used to assess protein expression levels of IDO1/2 and tryptophan 2,3-dioxygenase 2 (TDO2). In addition, the effects of notopterol on key proteins of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) and nuclear factor kappa B (NF-κB) signaling pathways were evaluated in a time- and dose-dependent manner. Direct inhibitory effects of notopterol on JAK2 activity were determined using a kinase activity assay. Furthermore, a co-culture system of CD8⁺ T cells and tumor cells was used to assess the impact of notopterol on T cell proliferation. The results demonstrated that, at non-cytotoxic concentrations, notopterol significantly inhibited kynurenine production in MDA-MB-231, 4T1, and HeLa cells, with IC₅₀ values of 6.05, 10.82, and 2.45 μmol/L, respectively. Notopterol downregulated the protein expression of IDO1/2 and TDO2 and suppressed IFN-γ-induced upregulation of IDO1 mRNA. Mechanistically, notopterol markedly inhibited the phosphorylation of JAK1/2, signal transducer and activator of transcription 1/3 (STAT1/3), IκB kinase alpha/beta (IKKα/β), inhibitor of nuclear factor kappa B alpha (IκBα) and NF-κB p65, thereby suppressing both JAK/STAT and NF-κB signaling pathways. In vitro kinase assays further confirmed that notopterol directly inhibited JAK2 containing both JH1 and JH2 domains (IC₅₀=2.39 μmol/L). Moreover, notopterol significantly promoted the proliferation of CD8⁺ T cells co-cultured with MDA-MB-231 or 4T1 cells. In conclusion, notopterol may exert immunomodulatory and antitumor effects in TNBC by targeting JAK2, thereby simultaneously suppressing the JAK/STAT and NF-κB signaling pathways, downregulating IDO1/2 and TDO2 expression, reducing kynurenine production, alleviating tryptophan metabolism-mediated immunosuppression, and enhancing CD8⁺ T cell proliferation. These findings provide new insights into the development of notopterol-based immunotherapeutic strategies for TNBC.