The effects of two kinds of Escherichia coli (E. coli) strain, wild-type E. coli W3110 and E. coli nir-Ptac, which has enhanced NO2 reduction activity, on oral CH4 emission and NO3 toxicity in NO3-treated sheep were assessed in a respiratory hood system in a 4×6 Youden square design. NO3 (1·3g NaNO3/kg0·75 body weight) and/or E. coli strains were delivered into the rumen through a fistula as a single dose 30min after the morning meal. Escherichia coli cells were inoculated for sheep to provide an initial E. coli cell density of optical density at 660nm of 2, which corresponded to 2×1010 cells/ml. The six treatments consisted of saline, E. coli W3110, E. coli nir-Ptac, NO3, NO3 plus E. coli W3110, and NO3 plus E. coli nir-Ptac. CH4 emission from sheep was reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac by 6% and 12%, respectively. NO3 markedly inhibited CH4 emission from sheep. Compared with sheep given NO3 alone, the inoculation of E. coli W3110 to NO3-infused sheep lessened ruminal and plasma toxic NO2 accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic NO2 accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation of E. coli nir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac. The inoculation of E. coli W3110 may abate NO3 toxicity when NO3 is used to inhibit CH4 emission from ruminants.