This study evaluated the effects of intraruminal administration of Megasphaera elsdenii on ruminal fermentation patterns, the profile of plasma metabolites, and milk yield and composition of mid-lactation dairy cows. Eight primiparous, ruminally cannulated Holstein cows were arranged in a paired 2×2 crossover design. Cows were randomly assigned to one of two treatments: 1) intraruminal inoculation of 35 ml suspension per day of M. elsdenii ATCC 25940 (MEGA), containing 108 cfu/ml of bacteria, dissolved in 35 ml of saline (0·15 m), or 2) carrier alone (35 ml saline; CTR). Both postprandial and preprandial rumen volatile fatty acids (VFA) and plasma metabolite measurements were analysed. Postprandial VFA patterns were affected the most, with butyrate (P<0·01) and valerate (P<0·01) proportions increasing, and acetate (P<0·01), isobutyrate (P=0·05) and isovalerate (P<0·01) decreasing in MEGA cows. Preprandial data measured at various days showed that MEGA dosage tended to increase the molar proportion of propionate (P=0·09) and lower the acetate to propionate ratio (P=0·07) in the rumen fluid. There was no effect of treatment on rumen pH and on the concentration of lactate in the rumen as well as on selected preprandial plasma metabolites. Postprandial plasma concentrations of cholesterol tended to increase (P=0·07) in MEGA cows compared with CTR. Concentrations of non-esterified fatty acids (NEFA) in the plasma were lower in MEGA cows after the morning feeding (P<0·01). Sampling hour also affected plasma NEFA in this study. Plasma β-hydroxybutyrate (BHBA) were not affected by the treatment (P>0·05); however, after the morning feeding BHBA concentration was increased in both groups of cows. Dry matter intake and milk yield and composition were not affected by treatment. In conclusion, results indicate that M. elsdenii has the potential to modulate the rumen fermentation profile in mid-lactation Holstein cows, but these effects were only slightly reflected in changes in plasma metabolites and milk composition.