【Objective】 The study aims to clone the fructokinase (FRK) gene of red pitaya (Hylocereus polyrhizus), detect its expression and enzyme activity, and analyze its physiological function on soluble sugar accumulation during fruit development. 【Methods】 HpFRK1 gene was cloned from ‘Zihonglong’ fruit, bioinformatics and subcellular localization analysis were carried out, gene expression was detected by qRT-PCR, recombinant protein was obtained by E.coli induction and enzyme activity was detected. 【Results】The open reading frame (ORF) of HpFRK1 was 993 bp in length, encoding 330 amino acids with a phosphofructokinase type B (pfkB) family domain. HpFRK1 has the closest genetic relationship with sugar beet BvFRK, both of which belong to cytoplasmic localization FRKs. HpFRK1 was expressed in different developmental stages of mature stems and fruits, the expression level of HpFRK1 was the highest at 20 days after flowering and the lowest at 30 days after flowering (fruit ripening), and gradually decreased with fruit development. Subcellular localization assay showed that HpFRK1 was mainly localized in the nucleus and cytoplasm. HpFRK1 recombinant protein specifically catalyzed fructose phosphorylation (Km=11.01 mmol/L). 【Conclusion】 HpFRK1 specifically catalyzes fructose phosphorylation in the cytoplasm and negatively regulates fruit soluble sugar accumulation during the development of red-fleshed pitaya fruit.