A study was conducted to determine the optimal standardized ileal digestible (SID) tryptophan:lysine (Trp:Lys) ratio in piglets challenged with Escherichia coli K88 (E. coli K88) and fed antibiotic-free diets. Thirty individually housed mixed-sex pigs (Duroc × [Yorkshire × Landrace]) with an initial BW of 6.41 ± 0.4 kg and weaned at 21 ± 1 d were randomly assigned to 5 dietary treatments with 6 replicates per treatment. Dietary treatments consisted of increasing levels of SID Trp:Lys ratios (16, 18, 20, 22 and 24%). Diets were corn-wheat-soybean meal-based with a constant SID Lys of 1.18% that was set to be the second limiting AA but adequate in other AA. Pigs had ad libitum access to feed and water for 13 d. Piglets were fed the experimental diets for 6 d and on d 7, they were orally challenged with 6 mL of E. coli K88 (2 × 10⁹ cfu/mL). Body weights and pen feed disappearance were recorded weekly to determine ADG, ADFI and G:F. On d 13, all pigs were euthanized to obtain ileal tissue samples to measure mRNA expression of tumor necrosis factor-α (TNF- α) and interleukin-10 (IL-10) using qRT-PCR. During the pre-challenge period, increasing dietary SID Trp:Lys increased (linear, P < 0.05) ADG (157, 162, 173, 179 and 201 g) and G:F (0.71, 0.73, 0.74, 0.81 and 0.84). During the post-challenge period, there was an increasing trend (linear, P = 0.076) in ADG (177, 180, 208, 210 and 213 g), whereas there was no effect on G:F (P > 0.10). The optimal SID Trp:Lys determined using the broken-line regression analysis for ADG and G:F in piglets subjected to E. coli K88 challenge was 21 and 24.2%, respectively. The expression of pro-inflammatory cytokine, TNF-α mRNA, linearly decreased (P = 0.10) with increasing SID Trp:Lys ratio. The expression of anti-inflammatory cytokine, IL-10 mRNA, increased (linear and quadratic, P < 0.01) with increasing SID Trp:Lys ratio. In conclusion, in antibiotic-free starter diets, an average optimal SID Trp:Lys of 22.6% optimized performance and immune status of piglets under E. coli challenge.