Glutamine is the preferred amino acid (AA) utilized by polymorphonuclear leukocytes (PMNL) during the inflammatory response. However, the effect of other AA on bovine PMNL response during inflammation and how this is altered by stage of lactation are currently unknown. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding glutamine) on AA profile, transcriptomic, and inflammatory function of PMNL from dairy cows in early and mid-lactation in vitro. Twenty Holstein dairy cows in early (n = 10; DIM = 17 ± 3.1) and mid-lactation (n = 10; DIM = 168 ± 14.8) were used for this study. PMNL were isolated and diluted using RPMI, containing basal concentrations of glucose (7.2 mM) and amino acids (3.1 mM). Working solutions of AA (0 mM or 4 mM of AA) and LPS (0 or 50 μg/mL) were added and tubes were incubated for 2 h at 37°C and 5% CO₂. Data were analyzed as a randomized block design. Stage of lactation did not alter PMNL responses in vitro. AA in combination with LPS increased (P ≤ 0.02) the concentration of alanine and methionine and tended (P < 0.10) to increase that of leucine, isoleucine, threonine, and phenylalanine. Regardless of LPS challenge, AA supplementation downregulated (P < 0.05) the expression of genes associated with inflammation such as NFKB1, IL10, IL1B, IL6, TNFA, LYZ, SOD2, and SLC2A3 but tended (P < 0.10) to increase the expression of TLR6, G6PD, LDHA, and PDHA1. Supplementation of AA reduced the concentration of TNF-α (104.0 vs. 34.9 ng/mL, P = 0.01) in medium but did not affect chemotaxis and phagocytic functions of PMNL. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain the lack of stage of lactation effects. This study identified AA other than glutamine that may alter PMNL response during inflammation in vitro that may lead to new avenues to improve immune response during inflammation in vivo.