Phoenixin (PNX) is a newly described peptide found in both neural and non-neural tissues. Until now, no attempts have been made to investigate the expression of PNX in the nervous system of animals other than laboratory rodents, in which an enzyme immunoassay revealed the highest quantity of the substance in the spinal cord. Since the domestic pig, due to its anatomical and histological resemblance to humans, is often used as an animal model in biomedical investigations, the present study was designed to examine PNX-immunoreactivity in the spinal cords of female pigs (n=5). The spinal cords were dissected and divided into the cervical, thoracic, lumbar, sacral and coccygeal segments, which were sectioned transversally into 10-μm-thick serial sections. The sections from each spinal cord segment were processed for double-labelling immunohistochemistry using antibodies against PNX in a mixture with those against calcitonin gene-related peptide (CGRP), substance P (SP) or choline acetyltransferase (CHAT). The PNX-immunoreactivity had a similar distribution in the grey matter of all the spinal cord sections examined and was mainly observed in varicose nerve fibres (NF) that formed a dense plexus in laminae I and II of the dorsal horn. Nearly all of the PNX-immunoreactive NF stained also for CGRP or SP and, interestingly, many of them were CHAT-positive. The present study has provided for the first time the detailed information on the arrangement and chemical features of nerve structures expressing PNX-immunoreactivity in the spinal cord of a large mammal. The exact function of PNX in the spinal cord is not known yet. However, the distribution pattern and immunohistochemical characteristics of PNX-IR NF clearly suggest that this peptite most likely plays a role in spinal noxious signalling.