Objective: To identify the proteins differently expressed between 3-week-old and adult mouse testes by label-free quantitative approach. Methods:Proteins were extracted respectively from 3-week-old and adult mouse testes,and digested by trypsin and subsequently analyzed by Nano LC-(liquid chromatography)-LTQ Orbitrap Mass Spectrometric Method. Using this method,the proteins were identified,while at the same time,the relative quantitation of protein expression was calculated according to peak intensity. Results:A grand total of 319 non-redundant proteins were identified,and 30 proteins among them were observed to exhibit significantly differential expression between 3-week-old and adult mouse testes(P < 0.05). These proteins primarily participate in morphogenesis,contraction,endocytosis and fertilization. These processes were tightly related with the spermiogenesis and sperm function. Conclusion:The label-free quatitative proteomic technique provided simple,high- throughput and low-cost measurement of protein expression levels. As such,it has a broad prospect in identifying related proteins involved in testis development,spermatogenesis and sperm function.