Tenocytes reside in relatively avascular tissue and are difficult to expand due to phenotype drift and functional loss. Thus low O-2 tension culture was employed to enhance the expansion capability. The results demonstrated that low O-2 tension (2% O-2) culture could significantly enhance the expansion of newborn pig tenocytes with 275-473% greater cell yield per cell passage that that of regular O-2 cultured (21% O-2) cells. Importantly, low O-2 culture did not change the gene expression of functional molecule such as collagens I and III, decorin, prolyl 4-hydroxylase (P4H), lysyl oxidase (LOX), TIMP-1 and TIMP-2, but could significantly down regulate the gene expression of MMP-1 and IL-6. In conclusion, low O-2 tension culture can significantly enhance the expansion capacity of tenocytes without affecting their phenotype and functions.