Abstract: Objective To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector. Methods The MAR of β-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method. Results The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron ( P<0.05). MAR did not improve transgene expression with reverse intron presence. Conclusion Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.