Arabidopsis bZIP11 is a transcription factor that modulates amino acid metabolism under high‐sucrose conditions. Expression of bZIP11 is downregulated in a sucrose‐dependent manner during translation. Previous in vivo studies have identified the second upstream open reading frame (uORF2) as an essential regulatory element for the sucrose‐dependent translational repression of bZIP11. However, it remains unclear how uORF2 represses bZIP11 expression under high‐sucrose conditions. Through biochemical experiments using cell‐free translation systems, we report on sucrose‐mediated ribosome stalling at the stop codon of uORF2. The C‐terminal 10 amino acids (29‐SFSVxFLxxLYYV‐41) of uORF2 are important for ribosome stalling. Our results demonstrate that uORF2 encodes a regulatory nascent peptide that functions to sense intracellular sucrose abundance. This is the first biochemical identification of the intracellular sucrose sensor.