Human CMP-N-acetylneuraminic acid (NeuAc) synthase (hCSS) and alpha 2,6-sialyltransferase (hST) participate in the sialylation of N-linked glycans in mammalian cells. hCSS synthesizes CMP-NeuAc, which hST uses as a donor substrate to transfer NeuAc to the terminal position of N-linked glycans. In plant cells, the presence of NeuAc has not yet been substantiated and the identification of the genes involved in the sialylation of N-glycan has not been carried out. In this study, we introduced hCSS and hST genes into suspension-cultured tobacco BY2 cells to provide the machinery for the sialylation pathway in plants. hCSS and hST stably expressed in the plant cells showed activity. In addition, CMP-NeuAc produced by hCSS in the transformed plant cells functioned as a donor substrate to hST. An in vitro coupled hCSS and hST reaction resulted in the production of mammalian-type sialoglycoproteins bearing terminal NeuAc residues. Furthermore, the results of the purification of the coupled-reaction products by Sambucus sieboldian lectin column chromatography and digestion with linkage-specific neuraminidase revealed that the modified terminal residue was alpha 2,6-linked NeuAc. Here, we demonstrate that the in vitro sialylation of N-linked glycans on mammalian proteins can be achieved using plant cell extracts stably expressing hCSS and hST, providing proof-of-principle that a sialylated human therapeutic protein can be produced in plants. Copyright (c) 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.