Supplementation of methionine (Met) to dairy cows is effective in the optimization of milk production and improvement of health. Isolated PMNL from multiparous Holstein cows were used to evaluate the effect of methionine supplementation on mRNA expression of galectin genes (LGALS-1, -2, -3, -4, -7, -8, -9, -12) in vitro and on the impact of stimulation with lipopolysaccharide (LPS). Galectins are important immunological mediators of homeostasis and disease regulation. PMNL was isolated from 10 Holstein cows and divided into 2 groups (Group 1: n = 5 and Group 2: n = 5). PMNL from Group 1 were incubated with 3 levels of lysine (Lys) to Met ratios of (3.6:1, 2.9:1, or 2.4:1). PMNL from Group 2 were also incubated with 3 levels of Lys to Met ratios of (3.6:1, 2.9:1, or 2.4:1) and 50 μg/mL of LPS. Cells were incubated at 37°C, with 5% CO₂ for 4 h. Met had no effect on LGALS expression in Group 1 cows. In Group 2 cows, however, LGALS9 expression was reduced after treatment with 2.4 Lys:Met. LGALS8 transcription was increased when PMNL were treated with 2.9 Lys:Met and LPS (P = 0.0005). LGALS8 was increased in PMNL treated with 3.6 Lys:Met(P = 0.05). LPS increased the expression of LGALS4 (P = 0.005) and tended to increase LGALS12 (0.08) in Group 2. These results shed light on the modulation of galectin expression in cows in response to methyl donors and LPS. The observed modulation of galectins expression by bacterial LPS and methionine may be exploited for the design of anti-inflammatory therapeutics.