Immune system activation increases nutrient and oxygen demand and could alter availability of amino acids (AA) for milk production. Given that the plasma free AA pool is small, changes in the concentrations could reflect changes in uptake or supply of AA by different organs. Plasma concentration of 3-methylhistidine (3-MH) is a good indicator for protein mobilization from muscle. Immune challenge can be modeled by administrating lipopolysaccharides (LPS) eliciting a well-characterized and robust immune response. Study objectives were to examine the impact of immune system activation by LPS on plasma free amino acid and 3-MH concentrations in lactating dairy cows. Twelve lactating Holstein cows were jugular catheterized and assigned to 1 of 2 bolus treatments: control (CON; 5 mL of saline, n = 6) and LPS (1.5 μg/kg of BW; Escherichia coli 055: B5; n = 6). Blood samples were obtained from the jugular vein immediately before, and 360 min after administrating saline or LPS. Protein was precipitated from plasma and individual amino acids and 3-MH in the filtrate were detected in an ion trap LC-MS system. Immunoactivation was successfully induced within 360 min as indicated by increased circulating levels of haptoglobin, serum amyloid A, and LPS-binding protein (all > 50%). The immunocativation was related to 80% and 7% decreases in milk yield and milk protein percentage, respectively and a 20% increase in milk urea nitrogen. Plasma concentration of arginine in LPS cows declined from baseline and was 60% less than that of CON cows (P = 0.07) at 360 min after LPS administration. Plasma concentrations of total branched-chain amino acids (BCAA, P = 0.07), and isoleucine alone (P = 0.07) were lower in LPS cows than CON cows throughout the LPS-challenge. Plasma concentration of the other essential amino acids, non-essential amino acids, or 3-MH did not change in response to the immunoactivation. The data indicate an acutely activated immune system could be associated with a lack of arginine and BCAA supply, while the contribution of AA mobilization from muscle is marginal.