Erythropoietin (EPO) is a glycoprotein hormone that regulates red blood cell formation in mammals. It also plays an important role in wound healing and the brain’s response to neuronal injury. Recombinant EPO has been produced in various hosts including Escherichia coli, insect cells, and yeast. Plant systems are also useful for the production of recombinant proteins. In this study, we used Nicotiana benthamiana for the production of recombinant human EPO (rhEPO) using an Agrobacterium-mediated transient expression system. We evaluated the effect of repeated agroinfiltration and Agrobacterium density on the rhEPO production. In addition, the rhEPO expression vector was coinfiltrated with a vector expressing cucumber mosaic virus suppressor 2b (CMV2b), which suppresses posttranscriptional gene silencing. We found that rhEPO expression was increased approximately 5.5-fold in N. benthamiana leaves coinfiltrated with CMV2b. In contrast, neither Agrobacterium density nor the number of infiltrations influenced rhEPO expression.