Liposomes are widely utilized in molecular biology and medicine as drug carriers. Here we report a new liposome-cell interaction through connexins. Connexin 43 (Cx43)-containing liposomes were prepared by using cell-free transcription/translation systems with plasmids encoding Cx43 in the presence of liposome. The expressed membrane protein, Cx43, was directly constituted to the liposome membrane upon in vitro synthesis, leading to pure membrane protein-containing liposomes. The hydrophilic dye calcein was efficiently transferred from Cx43-expressing liposomes to cultured cells (Cx43 expressing). The transfer is significantly blocked in the presence of gap junction inhibitor (18beta-glycyrrhetinic acid) and in the case of the other type of connexin (Cx32)-expressing cell. The results show that calcein entered the cell through connexin-mediated pathway. Cx43 liposomes containing a soluble NEMO-binding domain peptide suppressed the intracellular signaling cascade IL-1beta-induced NF-kappaB activation and cyclooxygenase-2 expression in Cx43-expressing cells, confirming effective peptide transfer into the cell. This is a new method for direct cytosolic delivery of hydrophilic molecules.