The objective of this study was to characterize the morphology and expression of the genes related to tight junction protein, cytokines, proliferation, and apoptosis in the gastrointestinal tracts (GIT) of lactating cows. Eighteen multiparous Holstein dairy cows were individually fed, and randomly assigned into 1 of 3 treatments. Isonitrogenous diets with forage-to-concentrate ratio at 45:55 contained similar concentrate and 15% corn silage, with 3 forage sources (DM basis): 23% alfalfa hay and 7% Chinese wild rye hay (AH); 30% corn stover (CS); and 30% rice straw (RS). The portion with length above 19 mm was the largest in RS, followed by AH, least in CS. After 14-wk feeding, all the cows were slaughtered. The digesta, tissue, and relevant epithelium of the GIT were collected immediately after dissection. The morphology of the GIT tissues was analyzed according to the hematoxylin-eosin stained method and the histomorphometric analysis was performed using Image J Plus software (Media Cybernetics, Bethesda, MD). The qRT-PCR reaction of the related genes in rumen epithelium was performed using the 2 × SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara, Otsu, Japan). The variance of the data was analyzed as a completely randomized design using PROC MIXED of SAS. The DM of rumen digesta was heavier in diet RS than in AH (P < 0.01). The thickness of ruminal mucosa was greater in RS than in AH (P < 0.05) or CS (P < 0.05), but the thickness of mucous epithelium was greater in AH (P < 0.05) or RS (P < 0.05) than in CS. The rumen butyrate concentration was greater in diet CS than RS (P < 0.01) or AH (P < 0.01), and had a negative correlation with the mRNA abundance of BCD (P = 0.01). Abundance of mRNA of IGF-1R was upregulated in AH compared with CS (P = 0.047). Abundance of mRNA of claudin-1 (P = 0.09), claudin-4 (P = 0.10), and ZO-1 (P = 0.07) tended to increase in AH compared with CS. The expression of gene related to cytokines showed no difference among the treatments. The expression of IGF-1 tended to increase in AH (P = 0.066) compared with CS. Our finding suggested that feeding of rice straw to dairy cow caused the change in ruminal mucosa mostly related to cell proliferation and apoptosis, which may be not only attributed to the physically effective fiber effects but also the butyrate factor.