Although the establishment of putative porcine embryonic stem cells (ESCs) has been reported, such cell lines quickly lose their self-renewal ability, as they easily differentiate or become extinct after only a limited number of passages in culture. ESC-like cells exhibiting self-renewal rather than pluripotency are considered to be a valuable resource in applications such as drug screening and toxicology testing in humans, livestock and veterinary medicine. Here, we report the generation of unique cell lines established from the inner cell mass (ICM) of porcine embryos by using inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1. These ICM-derived cell lines were initially cultured and passaged in conventional ES medium for human ESCs and showed porcine ESC-like morphology with alkaline phosphatase (AP) activity. After transfer to culture in ES medium containing inhibitors, the morphology of the colonies was dramatically changed, i.e., they were closely packed smooth-edged colonies with close cell-cell boundaries and showed the expression of undifferentiated markers including OCT4 (POU5F1) and NANOG. Notably, the self-renewal capacity and morphology of the cells were LIF-dependent, consistent with the expression of LIF receptors and phosphorylation of signal transducer and activator of transcription 3. To date, our established cell lines have been cultured continuously for over 100 passages without any overt morphological changes. Thus, the established cell lines reported here provide a new ESC-like cell culture system for use not only in the fields of veterinary medicine and livestock but also human medical research, since porcine physiology closely resembles that of humans.