3-Phosphoglycerate dehydrogenase (PHGDH) catalyzes the first rate-limiting step for the synthesis of glucose-derived serine by converting 3-phosphoglycerate (3-PG) to phosphohydroxypyruvate (p-Pyr), which has been reported to associate with tumorigenesis in many cancers. Iox A, a natural withanolide obtained from dietary tomatillo (Physalis ixocarpa), showed significant PHGDH inhibitory activity with an IC50 value of 1.66 ± 0.28 μM, and it was further confirmed to bind directly to PHGDH by the MST assay. Molecular docking demonstrated that Iox A coordinated at the allosteric site of PHGDH, which was consistent with the non-competitive kinetics. Meanwhile, Iox A selectively inhibited the proliferation of high PHGDH-expressing cancer cell lines (SW1990, MCF-7 and HeLa) and showed no obvious cytotoxicities on normal human cells (LO2, L929 and HPDE6-C7). In particular, Iox A showed a dose-dependent proapoptotic activity against SW1990 cells in a micromolar concentration as detected by flow cytometry and western blot analysis. DARTS and siRNA assays further demonstrated that Iox A directly targets at PHGDH to inhibit the proliferation of SW1990 cells. Furthermore, Iox A significantly inhibited the tumor growth in a SW1990 xenograft mouse model with low toxicities, suggesting its potential therapeutic application in pancreatic cancer treatment. Therefore, Iox A was identified as a novel natural PHGDH inhibitor with high targeting and low toxicities for the treatment of pancreatic cancers.