Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saponin of the root tuber of Pseudostellaria heterophylla on different developmental stages. Results showed that the primary structure and secondary structure of P. heterophylla adventitious root were similar to that of other herbaceous dicotyledons. 80% of mature root tuber were secondary xylem which consisted of most parenchyma cells and less vessels. The secondary phloem were composed of parenchyma cells too. The results of histochemical methods showed that saponin distributed in pericycle and parenchyma cells of primary phloem in the primary structure of root. In secondary structure and mature root tuber, saponin distributed in periderm and secondary vascular structure except cork layer and vessel. Besides, the colors of the secondary phloem were darker. The results of phytochemical test showed that the content of saponin in skin areas was higher than that in xylems in February and July. The result was consistent with that of histochemistry. The content of saponin in the head of the root was higher than that in the end of the root which was in turn higher than that in the middle part of the root. In the developmental process of root tuber, the content of saponin showed a dynamic tendency of high-low-high. This characteristics were related to the development of root system.