Monensin is a ionophor that selects against gram-negative rumen bacteria and increases the propionic acid proportion in the rumen, the main precursor of gluconeogenesis in the liver. Changes of rumen microbiota and fermentation pattern can also affect the permeability of epithelium. In previous studies, the inflammatory profile (leukocyte infiltration, cytokines and Ig content) of rumen fluids was suggested as a marker of these changes. Based on these findings, we investigated monensin effect on fermentation patterns and innate immune responses in the rumen of lactating cows. Forty-three dairy cows, 13 primiparous (PR) and 30 multiparous (ML), were allocated to 2 homogeneous groups: Kex received monensin by a controlled release capsule (approx. 335 mg/d, Kexxtone, Elanco Animal Health, UK) 21 d before expected calving and Ctrl (control). Fluid samples were collected with a rumen probe at 30 d in milk, 6 h after feed distribution. An aliquot was used for volatile fatty acids and ammonia analyses and one was used for flow cytometry and molecular assays. The prevalence of T and B lymphocytes and myeloid leukocyte cells was determined by monoclonal antibodies to CD3, surface IgM and an epitope expressed by the majority of monocytes and granulocytes in peripheral blood (IL-A24), respectively. Data were analyzed by one-way ANOVA, considering treatment and parity fixed effects. Kex cows showed a lower concentration of ammonia (58.7 vs 93.3 mg/L; P < 0.05), a higher level of propionate (24.5 vs 22.4 mol/100 mol; P < 0.10) and a reduced acetate:propionate ratio (2.5 vs 2.8; P < 0.10). Kex cows showed a lower B lymphocytes proportion compared with Ctrl (1.66 vs 2.50%; P < 0.05), in particular in PR cows. The T lymphocytes content of rumen fluid tended to be lower in Kex PR cows compared with Ctrl PR (P < 0.1), but no effect appeared in ML. These results confirmed monensin effects on rumen fermentations and a greater response of the immune system in the rumen of Ctrl cows. Moreover, the assessment of the rumen leukocytes activity under stressful physiological conditions could help to evaluate the impact of diet strategies on animal wellness.