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Dwarfism and semi-dwarfism are among the most valuable agronomic traits in crop breeding, which were adopted by the &#8220;Green Revolution&#8221;. Previously, we reported a novel semi-dwarf rice mutant (<i>oscyp96b4</i>) derived from the insertion of a single copy of <i>Dissociator (Ds)</i> transposon into the gene <i>OsCYP96B4</i>. However, the systems metabolic effect of the mutation is not well understood, which is important for understanding the gene function and developing new semi-dwarf mutants. Here, the metabolic phenotypes in the semi-dwarf mutant (M) and ectopic expression (ECE) rice line were compared to the wild-type (WT) rice, by using nuclear magnetic resonance (NMR) metabolomics and quantitative real-time polymerase chain reaction (qRT-PCR). Compared with WT, ECE of the <i>OsCYP96B4</i> gene resulted in significant increase of &#947;-aminobutyrate (GABA), glutamine, and alanine, but significant decrease of glutamate, aromatic and branched-chain amino acids, and some other amino acids. The ECE caused significant increase of monosaccharides (glucose, fructose), but significant decrease of disaccharide (sucrose); induced significant changes of metabolites involved in choline metabolism (phosphocholine, ethanolamine) and nucleotide metabolism (adenosine, adenosine monophosphate, uridine). These metabolic profile alterations were accompanied with changes in the gene expression levels of some related enzymes, involved in GABA shunt, glutamate and glutamine metabolism, choline metabolism, sucrose metabolism, glycolysis/gluconeogenesis pathway, tricarboxylic acid (TCA) cycle, nucleotide metabolism, and shikimate-mediated secondary metabolism. The semi-dwarf mutant showed corresponding but less pronounced changes, especially in the gene expression levels. It indicates that <i>OsCYP96B4</i> gene mutation in rice causes significant alteration in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and shikimate-mediated secondary metabolism. The present study will provide essential information for the <i>OsCYP96B4</i> gene function analysis and may serve as valuable reference data for the development of new semi-dwarf mutants.