Abstract To evaluate the effect of an Escherichia coli lipopolysaccharide (LPS) challenge on the digestible lysine (Lys) requirement for growing pigs, a nitrogen (N) balance assay was performed. Seventy-two castrated male pigs (19 ± 1.49 kg body weight [BW]) were allocated in a 2 × 6 factorial design composed of two immune activation states (control and LPS-challenged) and six dietary treatments with N levels of 0.94, 1.69, 2.09, 3.04, 3.23, and 3.97% N, as fed, where Lys was limiting, with six replicates and one pig per unit. The challenge consisted of an initial LPS dose of 30 μg/kg BW via intramuscular (IM) injection and a subsequent dose of 33.6 μg/kg BW after 48 h. The experimental period lasted 11 d and was composed of a 7-d adaptation and a subsequent 4-d sampling period in which N intake (NI), N excretion (NEX), and N deposition (ND) were evaluated. Inflammatory mediators and rectal temperature were assessed during the 4-d collection period. A three-way interaction (N levels × LPS challenge × time, P < 0.05) for IgG was observed. Additionally, two-way interactions (challenge × time, P < 0.05) were verified for IgA, ceruloplasmin, transferrin, haptoglobin, α-1-acid glycoprotein, total protein, and rectal temperature; and (N levels × time, P < 0.05) for transferrin, albumin, haptoglobin, total protein, and rectal temperature. LPS-challenged pigs showed lower (P < 0.05) feed intake. A two-way interaction (N levels × LPS challenge, P < 0.05) was observed for NI, NEX, and ND, with a clear dose–response (P < 0.05). LPS-challenged pigs showed lower NI and ND at 2.09% N and 1.69 to 3.97% N (P < 0.05), respectively, and higher NEX at 3.23% N (P < 0.05). The parameters obtained by a nonlinear model (N maintenance requirement, NMR and theoretical maximum N deposition, NDmaxT) were 152.9 and 197.1 mg/BWkg0.75/d for NMR, and 3,524.7 and 2,077.8 mg/BWkg0.75/d for NDmaxT, for control and LPS-challenged pigs, respectively. The estimated digestible Lys requirements were 1,994.83 and 949.16 mg/BWkg0.75/d for control and LPS-challenged pigs, respectively. The daily digestible Lys intakes required to achieve 0.68 and 0.54 times the NRmaxT value were 18.12 and 8.62 g/d, respectively, and the optimal dietary digestible Lys concentration may change depending on the feed intake levels. Based on the derived model parameters obtained in the N balance trial with lower cost and time, it was possible to differentiate the digestible Lys requirement for swine under challenging conditions.