The MGpUK‐5 cell line, transformed with a single‐chain urokinase‐type plasminogen activator (scu‐PA) minigene, generated mRNA transcripts and scu‐PA titers corresponding to 65% or 86% of the amount generated before serum‐free adaptation, despite significant loss of scu‐PA gene copies during adaptation to serum‐free culture. To further augment scu‐PA production, a culture strategy employing sodium butyrate was explored. In 60‐mL spinner flask cultures, sodium butyrate in the concentration range 1–10 mM allowed scu‐PA production 2‐ to 3‐fold higher than that in the negative control culture. Its productivity‐enhancing activity was dependent on cell density in a range of 1–5 × 106 cells/mL, generating 72,200 ± 8,100 IU/mL (480 ± 50 mg/L) in 60‐mL spinner flask cultures. To confirm this result, cells were grown to 4.4 × 106 cells/mL and treated with 5 mM sodium butyrate in a 2.5‐L perfusion culture. The scu‐PA titer increased more than 2‐fold, and specific production rate of scu‐PA increased 3‐fold by this treatment. Overall, this perfusion culture gave rise to 1.7 × 108 IU scu‐PA (1.1 g), comparable to total scu‐PA production in a batch butyrate‐treated culture performed at a 25‐L bioreactor scale (1.3–3.5 g). Our results suggest that sodium butyrate treatment on high‐density culture enables scu‐PA production in gram quantities.