The effects of subacute ruminal acidosis (SARA) on the electrophysiological and rumen epithelium barrier functions and its mechanism using dairy goats as a ruminant model were investigated. Eight ruminal cannulated female Sannan dairy goats of mid-lactating period (body weight 43.58 ± 2.77 kg) were selected. The experiment lasted 60 d, and divided equally into 4 periods. During each phase, animals were fed with 4 diets in which non-fiber carbohydrates/neutral detergent fiber ratios (NFC/NDF) were either 1.40, 1.78, 2.31, or 3.23 respectively. Ruminal fluid samples were collected for volatile fatty acids analyzed. Ruminal pH was measured continuously for 72 h. Ruminal tissue samples from the ventral sac were analyzed by Ussing chamber, where electrophysiological properties were determined in parallel with the permeability of marker molecules of different sizes, fluorescein isothiocyanate (FITC) and horseradish peroxidase(HRP), from luminal to apical side. Additionally, ruminal epithelial junction (TJs) proteins gene expressions were evaluated using quantitative real-time PCR and Western blot. The ruminal acetate concentration and Acetate/Propionate ratio (A/P) decreased, and the concentration of propionate, butyrate and total volatile fatty acids (VFA) increased with the increase of dietary NFC/NDF ratio. Rumen epithelial short- circuit current (Isc) and tissue conductance (Gt) increased in SARA goats compared with control goats, and the mucosal to serosal flux of HRP and FITC increased as well, indicating a higher permeability for SARA goats. SARA increased mRNA and protein expressions of Claudin-4 compared with control group. The mRNA and protein expressions of Claudin-1, Claudin-7, Occludin, ZO-1 and Connxin-43 in SARA group were lower than those in control. The mRNA and protein expressions of desmoglein-1 in SARA group were significantly lower than in the control group. Hence, dairy goats suffering from SARA could result in damaged ruminal epithelial barrier functions, resulting in increase of ruminal epithelial permeability.