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The <i>OsNRT2.3a</i> and <i>OsNRT2.3b</i> isoforms play important roles in the uptake and transport of nitrate during rice growth. However, it is unclear which cis-acting element controls the transcription of <i>OsNRT2.3</i> into these specific isoforms. In this study, we used a yeast one-hybrid assay to obtain the TATA-box binding protein OsTBP2.1, which binds to the TATA-box of <i>OsNRT2.3</i>, and verified its important role through transient expression and RNA-seq. We found that the TATA-box of <i>OsNRT2.3</i> mutants and binding protein OsTBP2.1 together increased the transcription ratio of <i>OsNRT2.3b</i> to <i>OsNRT2.3a</i>. The overexpression of <i>OsTBP2.1</i> promoted nitrogen uptake and increased rice yield compared with the wild-type; however, the <i>OsTBP2.1</i> T-DNA mutant lines exhibited the opposite trend. Detailed analyses demonstrated that the TATA-box was the key cis-regulatory element for <i>OsNRT2.3</i> to be transcribed into <i>OsNRT2.3a</i> and <i>OsNRT2.3b</i>. Additionally, this key cis-regulatory element, together with the binding protein OsTBP2.1, promoted the development of rice and increased grain yield.