This study was conducted to evaluate the effects of grass and flaxseed oil on weight gain, fecal pH, microflora and acid resistance of fecal E. coli. in Friesian-Holsteins. Fifteen Holsteins were fed either conventional diet (C, n=5), grass (40%) and concentrate supplemented with flaxseed oil (60%) (T1, n=5) or grass only (T2, n=5) for 141 days, and T1 and T2 groups were grazed on pasture except feeding time. Grabbing fecal samples and measuring body weight were carried out at test day 1, 60, 120, 141. To evaluate acid sensitivity of fecal E. coli, feces were incubated in pH 2.0 anaerobic solution during 1 h at room temperature and measured survival rate. In terms of diets, C group had the highest n-6:n-3 ratio with 21.93, followed by T1 group (0.92) and T2 group (0.51). C and T1 group had higher final weight, total gain weight and average daily gain than T2 animals (p<0.05). The fecal pH of C group was maintained much lower than T1 and T2 group since day 120 (p<0.05). Total anaerobe in feces of C group were higher than the other groups since day 120 (p<0.05), but coliform bacteria were not significantly different among groups. Yeast and mold in feces of C group were significantly higher than the other groups from day 60 to day 141 (p<0.05), which is considered to be relevant to yeast in the diets. After feces were incubated at strong acid, more fecal E. coli of C group (34.09%) survived than those of T1 group (3.79%) and T2 group (1.02%) (p<0.05). From those results, it was considered that feeding grass and flaxseed oil to Holsteins enables to change fecal microflora, increase acid sensitivity of fecal E. coli and contribute to stay healthy for animals. It would be necessary to develop various grass feed diets and their applications for producing healthy and high quality meat products.