Human embryonic kidney 293 (HEK293) cells with glycosylation machinery have emerged as an alternative host cell line for stable expression of therapeutic glycoproteins. To characterize dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification in HEK293 cells, an expression vector containing dhfr and monoclonal antibody (mAb) gene was transfected into dhfr-deficient HEK293 cells generated by knocking out dhfr and dhfrl1 in HEK293E cells. Due to the improved selection stringency, mAb-producing parental cell pools could be generated in the absence of MTX. When subjected to stepwise selection for increasing MTX concentrations such as 1, 10, and 100 nM, there was an increase in the specific mAb productivity (q(mAb)) of the parental cell pool upon DHFR/MTX-mediated gene amplification. High producing (HP) clones with a q(mAb) of more than 2-fold of the corresponding cell pool could be obtained using the limiting dilution method. The q(mAb) of most HP clones obtained from cell pools at elevated MTX concentrations significantly decreased during long-term culture (3 months) in the absence of selection pressure. However, some HP clones could maintain high q(mAb) during long-term culture. Taken together, a stable HP recombinant HEK293 cell line can be established using DHFR/MTX-mediated gene amplification together with dhfr(-) HEK293 host cells.