Agrobacterium‐mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield‐related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM⁻¹) and recombinant protein productivity per unit area‐time (g m⁻²/month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield‐related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m⁻² than at a low plant density of 100 plants m⁻². The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area‐time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium‐mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762–1770. © 2017 Wiley Periodicals, Inc.