Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by β- and ω-oxidation coupled to the Krebs cycle and β-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones.