This study investigated the effects of dietary 5-hydroxytryptophan (5-HTP) supplementation on growth performance, apparent total tract digestibility (ATTD), blood profile, intestinal morphology, transcriptomics, and microbial composition in weaned piglets. A total of twenty-four 28-day-old weaned piglets (Landrace × Large Yorkshire, 8.28 ± 1.09 kg) were randomly divided into 3 dietary treatments with 8 replicates. The dietary treatments include basal diet (CON), CON diet containing 250 or 500 mg/kg 5-HTP. The results revealed that supplementation with 250 mg/kg 5-HTP significantly increased (P < 0.05) the average daily gain (ADG) and resulted in a lower (P < 0.05) feed conversion ratio (FCR), while also decreased (P < 0.05) the diarrhea rate compared to the CON group. The ATTD of crude protein (CP) was lower in the 500 mg/kg group (P < 0.05) compared with the 250 mg/kg group. Furthermore, supplementation with 5-HTP led to significantly increased (P < 0.05) plasma albumin (ALB) and total protein (TP). In addition, supplementation with 5-HTP, particularly in the 250 mg/kg group, significantly increased (P < 0.05) serum serotonin (5-HT), growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels, and improved the ratio of villus height to crypt depth in the jejunum and ileum. The transcriptomic analysis revealed that the majority of differentially expressed genes (DEGs) induced by 5-HTP were related to digestion and immunity in the ileum, and 5-HTP enhanced (P < 0.05) intestinal glucose transporter 2 (GLUT2), solute carrier family 1 member 1 (SLC1A1) and solute carrier family 7 member 7 (SLC7A7) mRNA expression in weaned piglets. Furthermore, supplementation with 250 mg/kg 5-HTP increased (P < 0.05) abundance of Firmicutes, Actinobacteriota, Lachnospiracea, Ruminococcaceae and Megasphaera and decreased (P < 0.05) abundance of Spirochaetes and Treponema. Collectively, the study demonstrated that 5-HTP supplementation, particularly at 250 mg/kg, positively impacted growth performance, gut health, and microbiome composition in weaned piglets. These findings suggest the potential of using 5-HTP as a dietary supplement to enhance the health and productivity of weaned piglets.