Stressors such as weaning, social structure and environmental changes reduce piglet performance. Tryptophan (Trp) as a precursor of cerebral 5-hydroxytryptamine (5HT, serotonin) and melatonin may mitigate stress of animals. The photoperiod controls the transformation of Trp in its metabolites which in turn influence the consumption and welfare of piglets. This study aimed to evaluate the effects of light program and Trp levels on growth performance, blood glucose and plasma cortisol profile of piglets. Seventy-two 21-d-old piglets (initial BW = 6.6 ± 2.33 kg) were allotted to 4 treatments stratified by sex and initial BW with 6 pens/treatment and 3 piglets/pen. Data were analyzed as randomized complete block design in a 2 × 2 factorial arrangement (light programs: 12 or 23 h of light/d, and 2 digestible Trp levels: 2.6 or 5.2 g of L-Trp/kg of diet in pre-starter 1 from d 1 to d 14, and 2.4 or 4.8 g of L-Trp/kg of diet in pre-starter 2 from d 15 to d 24). The trial was carried out in a room with controlled temperature (26 and 23°C during the first 14 and during the last 10 d, respectively). Blood glucose was determined on d 0 and 24, and plasma cortisol on d 0 and 8. The baseline glucose and cortisol concentrations were used as a covariate adjustment in all analyses. No effects of light program and Trp levels interaction were observed on performance and blood glucose. From d 0 to 14 and 0 to 24, treatments did not influence ADG, ADFI and glucose, while G:F decreased (P < 0.05) from d 0 to 14 for pigs receiving 12 h of light/d compared with the ones receiving 23 h of light/d, but improvement was offset in the total period. Light program and Trp levels interaction was observed for plasma cortisol. Pigs receiving 23 h of light/d presented higher plasma cortisol (P < 0.05) than piglets receiving 12 h of light/d when fed diet with 2.6 g L-Trp/kg, however there was no difference on plasma cortisol (P > 0.05) in both light programs for pigs fed 5.2 g L-Trp/kg. In conclusion, higher levels of dietary Trp are recommended for piglets receiving light program of 23 h.