This study is to separate and identify the proteins in epididymal luminal fluid from the caput and cauda segments of normal mature rat’s epididymidis by two dimensional (2D)electrophoresis and mass spectrum. Twelve proteins were identified from 22 differential protein spots separated from the epididymal luminal fluid of caput and cauda epididymidis. Eleven of them were reported in epididymidis of different species of mammals except the peroxiredoxin6 (Prdx6). Prdx6 was a novel epididymal protein presented in the luminal fluid of caput and cauda epididymidis. Prdx6 was further studied for expression and distribution in rat’s epididymidis with RTPCR, Western blot and immunohistochemistry. Results suggested that Prdx6 protein may play a role in sperm maturation, storage and protection in epididymidis. The concrete mechanism of Prdx6 is worth further investigating.