This study was conducted to analyze the effects of 5-HMF (5-hydroxymethylfurfural) on the growth performance, intestinal histology, serum biochemical characteristics, and gastrointestinal microecology of juvenile yellowtail kingfish. A total of 360 6-month-old yellowtail kingfish juveniles with an initial mean body mass of (82.33±2.75) g were randomly divided into four groups (M0 group, M1 group, M2 group, and M3 group) with three replicates per group and 30 fish assigned in each replicate. The M0 group was the control group and was fed the basic diet, whereas the M1, M2, and M3 groups were fed the experimental diets (0.25% 5-HMF, 0.5% 5-HMF, and 1% 5-HMF, respectively). The experiment lasted for six weeks. The results showed that the proliferation experiment of Caco-2 cells (human colorectal adenocarcinoma cells) in vitro showed that the concentration of 5-HMF used in this study was within the safe range. The weight gain rate and specific growth rate of the M2 and M3 groups were significantly higher than those of the M0 group (P＜0.05) and the plumpness of the M2 group was significantly higher than that of the M0 group (P＜0.05). The muscle layer thickness and villus height of the M2 group were significantly higher than those of the M0 group (P＜0.05). The activities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and catalase (CAT) in the serum of M2 and M3 groups were significantly higher than those in the serum of M0 group (P＜0.05), while the level of malondialdehyde (MAD) was significantly lower (P＜0.05). The serum levels of immunoglobulin M (IgM) and alkaline phosphatase (AKP) in M2 and M3 groups were significantly higher than those in the M0 group (P＜0.05). The activities of lysozyme (LZM) and acid phosphatase (ACP) in the M2 group were significantly higher than those in the M0 group (P＜0.05). In the M0 group, the Chao1 index of pyloric caecum microbiota was significantly higher than that of the M1 group (P＜0.05), while the Shannon index of intestinal microbiota in the M2 group was significantly higher than that in the M0 and M1 groups (P＜0.05). The results of this study showed that the addition of 0.5% 5-HMF to feed could improve the growth performance and promote serum immune levels and antioxidant capacity of yellowtail kingfish. 5-HMF may have played a role in optimizing and regulating the intestinal morphology and microbial community structure of juvenile yellowtail kingfish. This provides theoretical support for the development of healthy breeding technologies and green feed additives for yellowtail kingfish.