Abstract: This study was supposed to evaluate the effects of three non-ionic surfactants (NIS) (Tween 40, 60 and 80) on nutrient digestibility and rumen fermentation. Four Chinese Merino rams, fitted with ruminal and duodenal cannulas, were used in a 4x4 Latin square designed experiment. The four treatments were the basal diet (the control) and the basal diet supplemented with 10 g day-1 Tween 40, 60 or 80, respectively. Results showed that there were no differences among the four treatments in nutrient intakes. When compared to the control, feeding NIS had no effect on ruminal or total tract digestibility of Organic Matter (OM), Crude Protein (CP), cellulose and hemicellulose. Sheep consuming Tween 60 or 80 tended to have higher apparent ruminal digestion of Dry Matter (DM) (p = 0.074). On rumen fermentation characteristics, ruminal pH and protozoa counts were not affected by NIS supplementation. However, carboxymethylcellulase activity was improved by Tween 60 and 80 (p<0.01). Sheep feed Tween 40, 60 or 80 resulted in lower (p<0.05) ruminal ammonia nitrogen (NH3-N) and higher (p<0.01) total Volatile Fatty Acid (VFA) concentrations. In terms of individual VFA, including Tween 60 or 80 in the diet significantly improved concentrations of acetate (p<0.01), propionate (p<0.01) and butyrate (p<0.05) in ruminal fluid but did not alter the ratio of acetate to propionate. It was concluded that Tween 60 and 80 reduced rumen NH3-N concentration and increased carboxymethylcellulase activity and total VFA, as well as acetate, propionate and butyrate concentrations but had no effect on nutrient intakes and digestion. It is inferred that potential uses of the Tween 60 and 80 as feed additives for sheep to optimize ruminal fermentation.