Substantial efforts have been made to manipulate ruminal environment in a hope to enhance ruminal fermentation efficiency for better ruminant productivity. Some of examples are methane inhibitors, antibiotics, microbial enzymes, fatty acids and/or lipid feeding, buffering agents, ionophores and probiotics. Of these efforts, the non-ionic surfactant (NIS) has been known for its stimulation to release enzymes from a range of anaerobic microbes. A series of studies were conducted 1) to evaluate NIS diluted with water and ethanol on in vitro ruminal fermentation and 2) to determine the influence of diluted NIS on digestibility of feedstuffs. In first experiment (Exp. 1), NIS was diluted with water or ethanol to measure its effects on in vitro microbial growth, ruminal enzyme activities and gas production by mixed ruminal microbial culture. The NIS was diluted with water or ethanol separately in a 1:5 ratio (w/v). Water and ethanol-diluted NIS with wheat flour were added with rice straw-based mixed ruminal microbial cultures at the rate of 2 ㎎ NIS/16 ㎖ McDougall buffer plus 4 ml ruminal fluid solution. The mixed ruminal microbial culture was run without any NIS addition as control. Addition of NIS either diluted with water or ethanol has significantly reduced the gas production in mixed ruminal microbial culture at 12 and 24 h of incubation. At 48 h post incubation, gas production was the highest with the addition of ethanol diluted NIS followed by water-diluted NIS and control. Carboxy methyl cellulase activity in rice straw-based mixed ruminal bacterial culture was significantly higher with the addition of ethanol-diluted NIS compared with the addition of water-diluted NIS and control at 24 and 72 h post incubation. In second in vitro experiment (Exp. 2), effects of addition of ethanol diluted NIS on dry matter (DM) digestibility of alfalfa hay, gas production, pH and cellular growth in mixed ruminal microbial culture were examined. Alfalfa hay based mixed ruminal microbial culture without any NIS addition was run as a control. The pH of mixed ruminal microbial culture was significantly lower than control at all post incubation sampling hours. In vitro DM digestibility of alfalfa hay was significantly higher with the addition of NIS compared with control. Gas production was significantly less with NIS addition compared with control at all post incubation sampling hours. Microbial growth in mixed ruminal microbial culture was significantly increased with the addition of NIS compared to control.