Tryptophan (Trp), the rate-limiting substrate of serotonin [5-hydroxytryptoamine (5-HT)] synthesis in the brain, competes with large neutral amino acids (LNAA) to cross the blood-brain barrier. This study was designed to evaluate the effect of L-Trp supplementation on nursery pigs experiencing social-mixing stress and fed diets varying in LNAA concentrations. Forty-eight individually housed barrows at 6 wk of age were randomly allotted to 4 dietary treatments based on a 2 × 2 factorial arrangement, with L-Trp supplementation (0 or 0.6%) and LNAA concentrations (4.5 or 3.8%) as the 2 main factors. Pigs were fed the diets for 7 d. On d 4, pigs within a treatment were paired in a new pen to create social-mixing stress and behavior was recorded for 24 h. Body weight was measured on d 0, 4, 5, and 7. Saliva and blood were collected on d 4 and 7. On d 7, pigs were killed to obtain hypothalami. During the entire period excluding the mixing day (d 5), L-Trp supplementation improved (P < 0.01) feed efficiency of pigs and lowering the LNAA further enhanced (P < 0.05) the effects of L-Trp. Supplementation of 0.6% L-Trp increased (P < 0.001) hypothalamic 5-HT and 5-hydroxyindoleacetic acid. The salivary cortisol concentration was reduced (P < 0.05) by lowering the LNAA. Collectively, lowering the LNAA further enhanced the improvement of feed efficiency by L-Trp supplementation of nursery pigs under social-mixing stress in association with reduced stress hormones, indicating that reducing LNAA in the diet can facilitate the effect of L-Trp on the stress response of pigs.