In this study, we examined the effects of rumen-protected L-tryptophan supplementation on the productivity and physiological metabolic indicators in lactating Holstein cows under heat stress conditions. The study involved eight early lactating Holstein cows (days in milk = 40 ± 9 days; milk yield 30 ± 1.5 kg/day; parity 1.09 ± 0.05, p < 0.05), four cows per experiment, with environmentally controlled chambers. In each experiment, two distinct heat stress conditions were created: a low-temperature and low-humidity (LTLH) condition at 25 °C with 35-50% humidity and a high-temperature and high-humidity (HTHH) condition at 31 °C with 80-95% humidity. During the adaptation phase, the cows were subjected to LTLH and HTHH conditions for 3 days. This was followed by a 4-day heat stress phase and then by a 7-day phase of heat stress, which were complemented by supplementation with rumen-protected L-tryptophan (ACT). The findings revealed that supplementation with ACT increased dry matter intake as well as milk yield and protein and decreased water intake, heart rate, and rectal temperature in the HTHH group (p < 0.05). For plateletcrit (PCT, p = 0.0600), the eosinophil percentage (EOS, p = 0.0880) showed a tendency to be lower, while the monocyte (MONO) and large unstained cells (LUC) amounts were increased in both groups (p < 0.05). Albumin and glucose levels were lower in the HTHH group (p < 0.05). The gene expressions of heat shock proteins 70 and 90 in the peripheral blood mononuclear cells were higher in the ACT group (HTHH, p < 0.05). These results suggest that ACT supplementation improved productivity, physiological indicators, blood characteristics, and gene expression in the peripheral blood mononuclear cells of early lactating Holstein cows under heat-stress conditions. In particular, ACT supplementation objectively relieved stress in these animals, suggesting that L-tryptophan has potential as a viable solution for combating heat-stress-induced effects on the cattle in dairy farming.