Ribokinase (RK), a member of the phosphofructokinase B (PfkB) family, catalyzes the ATP-dependent phosphorylation of d-ribose to ribose-5-phosphate, a step essential for nucleotide biosynthesis and pentose phosphate pathway flux. Because these pathways support redox balance, energy metabolism, and cell proliferation, RK is relevant to cancer and ischemic injury. A conserved feature of PfkB enzymes is the β-clasp dimerization motif, an interlocking β-strand interface between protomers. However, its quantitative contribution to human RK stability and catalysis remains unclear. Here, we define the structural and functional role of the RK β-clasp using structure-guided mutagenesis and integrated functional analyses. The β-clasp contains two interaction layers: (1) a β3a–β7 backbone hydrogen-bond networkthat forms the core dimer scaffold and (2) a surrounding network of polar and hydrophobic interactions that reinforce the interface. Alanine substitutions disrupting individual polar interactions were largely tolerated, preserving dimerization, near-wild-type activity, and thermal stability. However, disruption of the V122/V124-centered hydrophobic core reduced catalytic turnover with minimal effects on substrate affinity, indicating that the β-clasp primarily supports catalytic competence rather than substrate recognition. Progressive β3a deletions destabilized the dimer in a graded manner, yielding inactive, predominantly monomeric variants with noncooperative biphasic thermal unfolding transitions, although far-UV circular dichroism confirmed retention of the α/β fold. Molecular dynamics simulations showed that disruptive variants increased β-clasp flexibility, weakened the Asp27-centered ribose-binding network, and reduced ribose retention. Together, these findings identify the β-clasp as a conformational hub coupling dimerization to lid positioning and productive phosphoryl transfer, establishing it as a potential allosteric target for regulation.