The objective of this study was to characterize the immunohistochemical localization of plasma membrane connexin 43 in the rumen of sheep after changing the diet from hay (ad libitum) to a mixed hay/concentrate diet. A total of 24 sheep were fed mixed hay/concentrate for different periods ranging from 0 weeks (control; hay ad libitum) to 12 weeks (1-1.5 kg hay plus 780 g concentrate per day in two equal portions). Using immunohistochemical technique the present study confirmed the existence of plasma membrane connexin 43 in the sheep rumen epithelium. Plasma membrane connexin 43 immunostaining was most intense at the stratum basale and stratum spinosum (suprabasal layer) and decreased iron intensity through stratum spinosum (superficial layers) to stratum granulosum. Meanwhile, stratum corneum was negative. The reaction around the cells gave a syncitial appearance with more apical-immunostaining concentration. Moreover, the present study confirmed a significant effect of concentrate diet on the immunoreactivity of plasma membrane connexin 43 in the rumen of sheep. A very strong degree of antibody reaction was seen in 4 to 12 weeks concentrate-fed groups.