Study objectives were to evaluate effects of an endomicrobial feed supplement (EFS) (Galaxis; ASCUS Biosciences Inc., San Diego, California) comprised of 2 native rumen microbes on production parameters in mid-lactation dairy cows. Forty-seven lactating primiparous and multiparous Holstein cows (629 ± 9 kg BW; parity 1.9 ± 0.1; 119 ± 6 DIM; 45.11 ± 0.92 and 52.73 ± 0.87 kg/d MY for primiparous and multiparous, respectively) were enrolled in a study containing 2 experimental periods (P), during both periods cows were fed the same base TMR ad libitum once daily. During P1 (7 d) baseline data were obtained for covariate analysis. At the beginning of P2 (60 d), cows were randomly assigned to 1 of 2 dietary treatments: 1) a control diet (CON; base TMR; n = 24) or 2) a control diet supplemented with 5 g/d of EFS (EFS; n = 24). Overall, EFS had no effect on milk yield, ECM, DMI, BW, or BCS. However, EFS tended to increase feed efficiency (FE) and total milk solids content when compared with CON cows (2.2 and 1.1%, respectively; P = 0.08) and numerically increased protein content compared with CON cows (2.99 vs. 2.96%; P = 0.13). Cows from the original data set were retrospectively categorized into 2 milk production groups (MPG) within parity and classified as MPG1 (i.e., lower than median) and MPG2 (i.e., higher than median) based upon median P1 ECM (43.2 and 51.8 kg/d for primiparous and multiparous cows, respectively). EFS supplemented MPG1 cows had increased ECM (4.2%) and FE (2.8%) compared with CON cows (P = 0.02 and 0.05, respectively). There was a weak tendency for log somatic cell count to be increased in MPG1 EFS cows as compared with MPG1 CON cows (P = 0.12). There were no effects of EFS on production variables for MPG2 cows. In summary, EFS supplementation had little overall effects on production performance when all animals were analyzed. However, EFS supplementation improved milk production efficiency in lower producing primiparous (<43.2 kg/d) and multiparous (<51.8 kg/d) animals. Further investigation is needed to understand production level response differences.