ABSTRACTHydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R‐selective HNL enzyme containing an α/β‐hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1‐C2 bond and the rotation of the generated cyanide ion (CN−) follow, afterwards, CN− abstracts a proton from His236 via Ser81. The C1‐C2 bond cleavage and the protonation of CN− correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol−1), suggesting that both of the two processes contribute a lot to rate‐limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1‐C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol−1. The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN− is stabilized by the hydroxyl group of Ser81 and the main‐chain NH‐groups of Ala13 and Phe82. Proteins 2015; 83:66–77. © 2014 Wiley Periodicals, Inc.