The effects of different level of rumen-protected L-tryptophan (RPL-T) supplementation on starch digestion, melatonin (MEL) and gastrointestinal (GI) hormones secretion in Holstein steers were evaluated. Four Holstein steers (201 ± 24 kg) were employed in a 4×4 Latin square design. The dietary treatments were the control (basal diet) and RPL-T groups of basal diet + 191.1 mg/kg body weight (BW), basal diet + 95.6 mg/kg BW, and basal diet + 19.1 mg/kg BW groups. Blood samples were collected to measure blood hormones on day 0, 1, 3, and 5 of the experiment to study serum MEL and GI tract and duodenal starch degradability. The design was 4 × 4 Latin square and the data were analyzed using the ANOVA procedure by SPSS. The D-glucose content in the RPL-T treatment groups was significantly reduced (