Objective To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated chromosome inactivation. Results Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in cis on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in cis on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation. Conclusions We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins.