The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (Delta D2, Delta D4, and Delta D2 Delta D4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the Delta D2 Delta D4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than Delta D2, Delta D4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in Delta D2 Delta D4 plants than in Delta D2, Delta D4, and wild-type plants. These findings demonstrate that Delta D2 Delta D4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.