The rate-limiting serine biogenesis enzyme PHGDH is overexpressed in cancers. Both serine withdrawal and genetic/pharmacological inhibition of PHGDH have demonstrated promising tumor-suppressing activities. However, the enzyme properties of PHGDH are not well understood and the discovery of PHGDH inhibitors is still in its infancy. Here, oridonin was identified from a natural product library as a new PHGDH inhibitor. The crystal structure of PHGDH in complex with oridonin revealed a new allosteric site. The binding of oridonin to this site reduced the activity of the enzyme by relocating R54, a residue involved in substrate binding. Mutagenesis studies showed that PHGDH activity was very sensitive to cysteine mutations, especially those in the substrate binding domain. Conjugation of oridonin and other reported covalent PHGDH inhibitors to these sites will therefore inhibit PHGDH. In addition to being inhibited enzymatically, PHGDH can also be inhibited by protein aggregation and proteasome-mediated degradation. Several tested PHGDH cancer mutants showed altered enzymatic activity, which can be explained by protein structure and stability. Overall, the above studies present new biophysical and biochemical insights into PHGDH and may facilitate the future design of PHGDH inhibitors.