Ophiopogonis Radix (Maidong in Chinese), the tuberous root of Ophiopogon japonicus, is a commonly used tonic Chinese medicines. Through years of studies, it was found that major compounds isolated in Ophiopogonis Radix include steroidal saponins, homoisoflavonoids and polysaccharides, which possess multiple pharmacological activities such as cardiovascular protection, anti-inflammation, anticancer, immunomodulation, and anti-diabetes [1]. In China, Ophiopogonis Radix is mainly produced in Sichuan and Zhejiang provinces, where it is called Chuanmaidong (CMD) or Zhemaidong (ZMD) accordingly. Traditionally, the quality of ZMD is considered the best, but the production of CMD is the biggest at present. Therefore, it is important to compare the chemical characteristics and biological activities of Ophiopogonis Radix from different regions to ensure the safety and efficacy for medication. In this study, a pressurized liquid extraction and HPLC-DAD-ELSD method was developed for simultaneous determination of 3 steroidal saponins (ophiopojaponin C, ophiopogonin D, and ophiopognin D') 1 sapogenin (ruscogenin) and 4 homoisoflavonoids (methylophiopogonone A, methylophiopogonone B, methylophiopogonanone A, and methylophiopogonanone B) in CMD and ZMD. The results showed that Ophiopogonis Radix from different regions had very different chemical constitution and content. ZMD had much higher content of homoisoflavonoids, while the contents of ophioponin D and ophiopogonin D' were higher in CMD. Then Ophiopogonis Radix from different regions were well differentiated by principal component analysis (PCA) according to the content of the tested compounds. Furthermore, cytotoxic and neuro-protective activities of the 8 compounds were investigated. Among them, ophiopognin D' had the most prominent cytotoxic effect against ovarian cancer A2780 cells with an IC₅₀ value of 0.89±0.64μM, and the 4 homoisoflavonoids possessed similar cytotoxic activity with the IC₅₀ values of less than 10 μM. For neuroprotective activity, except ophiopogonin D and methylophiopogonanone A, other 6 investigated compounds could improve PC12 cell damage induced by 6-OHDA, t-BHP or L-Glu at different concentrations. The present results suggest that CMD and ZMD had significantly different chemical characteristics and biological activities, careful evaluation and consideration should be taken into account in clinical applications.