| Summary: | (1) Background: Exercise is effective in promoting and maintaining bone mass. The aim of this study was to detect the exercise-induced metabolic changes in bone tissue of zebrafish. (2) Methods: Thirty-eight zebrafish (Danio rerio, six months old) were analyzed. The exercise group (<i>n</i> = 19) received 8 weeks of counter-current swimming training. The control group (<i>n</i> = 19) was not subjected to exercise. Mineralization was quantified, and alkaline phosphatase (Alp) and anti-tartrate acid phosphatase (Trap) activities were estimated (<i>n</i> = 12). The metabolomics (<i>n</i> = 12) and transcriptomics (<i>n</i> = 14) data of bone tissue were used for the integration analyses. (3) Results: The results showed that the exercise training improved the bone mineralization of zebrafish, e.g., the exercise group (5.74 × 10<sup>4</sup> ± 7.63 × 10<sup>3</sup>) had a higher mean optical density than the control group (5.26 × 10<sup>4</sup> ± 8.56 × 10<sup>3</sup>, <i>p</i> = 0.046) for the caudal vertebrae. The amount of mineralized matrix in scales of the exercised zebrafish was also higher (0.156 ± 0.012 vs. 0.102 ± 0.003, <i>p</i> = 0.005). Both histological staining and biochemical analysis revealed increased Alp activity (0.81 ± 0.26 vs. 0.76 ± 0.01, <i>p</i> = 0.002) and decreased Trap activity (1.34 ± 0.01 vs. 1.36 ± 0.01, <i>p</i> = 0.005) in the exercise group. A total of 103 different metabolites (DMs, VIP ≥ 1, fold change (FC) ≥ 1.20 or ≤0.83, <i>p</i> < 0.050) were identified. Alanine, aspartate and glutamate metabolism, β-alanine metabolism, pyrimidine metabolism, and pantothenate and CoA biosynthesis were the significantly enriched metabolic pathways (<i>p</i> < 0.050). A total of 35 genes (<i>q</i> ≤ 0.050 (BH), |Log2FC| ≥ 0.5) were coenriched with the 103 DMs in the four identified pathways. Protein–protein interaction network analysis of the 35 genes showed that <i>entpd3</i>, <i>entpd1</i>, and <i>cmpk2</i> were the core genes. (4) Conclusions: The results of this study suggest that alanine, aspartate and glutamate metabolism, β-alanine metabolism, pyrimidine metabolism, and pantothenate and CoA biosynthesis contributed to exercise-induced improvements in bone mass.
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