A high performance liquid chromatographic method followed protein precipitation and liquid-liquid extraction respectively was developed and validated for the determination of MCC-555, a novel insulin sensitizer, in biological matrix. The calibration curves were linear in each sample range and both inter-day and intra-day RSD were below 15%. The method has been successfully used to support the pharmacokinetics and toxicokinetics study of MCC-555 in beagle dogs and rats. The absorption, distribution, metabolism and excretion of MCC-555 in animal were studied as well as in vitro binding of MCC-555 to human plasma proteins. The possible metabolites and path of MCC-555 metabolism in vivo and in vitro were supposed. And pathological histology observation of MCC-555 was also analyzed.