To determine the effects of the Sus scrofa matrix attachment region (SusMAR) on transgene expression in HEK293T cells. Three expression vectors with the MAR at different sites in the PiggyBac (PB) transposon vector backbone were compared: two MARs flanking the beta-galactosidase (beta-gal) expression cassette, and one at the upstream or downstream site. Bos taurus MAR (BosMAR) and a beta-gal expression cassette without MARs were the positive and negative controls, respectively. Compared to the control, beta-gal activity of all SusMAR and BosMAR vectors was significantly improved in the presence of PB transposase (PBase). However, only the downstream SusMAR and upstream BosMAR vectors showed increased expression in the absence of PBase. Expression was significantly increased in all vectors with the PBase group compared to those without the PBase group. Gene copy numbers were not increased compared to the negative control. SusMAR enhanced recombinant gene expression levels and stability in HEK293T cells, was not increase transgene copy number. These results could facilitate the development of vectors for stable production of therapeutic proteins.