Class D β-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in Gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae . Carbapenemase activity is an unusual feature among class D β-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands β5 and β6 (the β5–β6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D β-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the β5–β6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10–derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted β5–β6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the β5–β6 loop in the carbapenemase activity of class D β-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.