The production of estrogen receptors (ER) in cultured insect cells is advantageous because these cells are relatively easy to culture and they perform post-translation modifications necessary for protein stability and function. There are three options for protein expression in insect cells: transient transfection, lytic baculovirus infection, or transfection followed by selection to create stable cell lines. Stable transfection has been promoted to be advantageous for the production of recombinant proteins because no re-infection is required, which might provide better lot-to-lot reproducibility in protein production. In this paper, we demonstrate that lytic baculovirus infection of Sf21 cells yields approximately tenfold more bioactive ERbeta than cells stably transformed with pIZ/V5-His plasmid under OpIE2 promoter. We provide the first evidence that stable expression of recombinant human ERbeta decreases the proliferation of Sf21 cells by inhibition of cell replication in a ligand-independent manner. These results mirror findings in breast cancer cells showing that an increase in ERbeta expression decreases cell proliferation. We conclude that baculovirus infection of Sf21 cells is better for human ERbeta production than stable-transformation of Sf21 cells.