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The human membrane‐bound α/β‐hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2‐arachidonylglycerol (2‐AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2‐AG‐hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane‐bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2‐AG and esters of medium‐length fatty acids. PA2949 in the presence of n‐octyl β‐d‐glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6‐like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult‐to‐crystallize proteins. Homologous expression of the membrane‐bound esterase PA2949 from Pseudomonas aeruginosa PA01 and the purification of detergent‐solubilized enzyme resulted in stable PA2949 protein that crystallized. The crystals obtained were used for X‐ray analysis and diffracted to a resolution of 2.74 Å. [ABSTRACT FROM AUTHOR]