Red clover (Trifoliumpratense L.) as a dietary supplement is currently used to treat menopausal symptoms because of its high concentrations of mildly estrogenic isoflavone. The application rate of N and P fertilizer has a huge influence on the isoflavone content (IC) in red clover. Trifolium pratense ’Minshan’ is a unique plant resource in the alpine pasture region of northwest China. It plays a very important role in livestock production and the pharmaceutical industry because of its high nutritional value and IC. To date, little information exists on the selection of optimal N and P application rates to maximize the IC in red clover. Therefore, a 3-yr study was conducted to define the optimum N and P application rate to achieve a high IC in ’Minshan’. The results showed that topdressing 50 kg N ha-1 yr-1 with 52-105 kg P ha-1 yr-1 could produce a high level of IC at the branching, flower bud, early flowering, full flowering, pod setting, and mature stages in red clover. Second-year crops produced higher IC than did third-year crops. The IC was highest during the vegetative growing period and started to decline following the reproductive development of the plant until the seeds matured.