ScRBK1 is a key enzyme responsible for the ATP-dependent phosphorylation of d-ribose, and plays a crucial role in many metabolic processes in S. cerevisiae. Herein, we demonstrate that ScRBK1 enzymatic activity is independently stimulated by monovalent cation and inorganic phosphate ion. We determined the crystal structures of ScRBK1-ADP and ScRBK1-d-ribose complexes. Each ScRBK1 monomer consists of a small lid domain and a large catalytic α/β domain, exhibiting the typical structural characteristics of the carbohydrate kinase PfkB family. We mapped the critical interactions of ScRBK1 with ADP and d-ribose, as well as the activator monovalent cation. We identified key residues contributing to the enzymatic activity of ScRBK1, and elucidated the molecular mechanism underlying inorganic phosphate ion-dependent activation. Furthermore, our structural analyses highlighted the structural features, and interaction modes with both nucleotide and d-ribose substrates in HsRBK. Collectively, our study provides comprehensive structural and functional insights into the activation mechanisms of ScRBK1 by inorganic phosphate ion and monovalent cation, as well as the molecular mechanism of d-ribose phosphorylation, and reveals that HsRBK shares a common catalytic mechanism with RBK family members.