Density functional theory (DFT) calculations have been performed to investigate the mechanism and origin of the enantio- and regioselectivity of Rh-catalyzed desymmetric [2 + 2 + 2] cycloaddition of enynes with terminal alkynes. The computational results show that the catalytic cycle involves oxidative cyclization of cyclohexadienone alkynes (rate- and enantioselectivity-determining step), alkyne insertion (regioselectivity-determining step), and reductive elimination/catalyst regeneration. Natural population analysis and independent gradient model analysis show that for para-electron donating group-substituted aryl alkynes, the charge distribution of alkynes is the dominant factor controlling the regioselectivity, while for strong electron withdrawing group-substituted aryl alkynes, the regioselectivity is controlled by enhanced π–π stacking interactions between the phenyl group of the aromatic alkyne moiety and the phenyl group of the ligand. Distortion/interaction analysis shows that the interaction energy between the rhodacycle complex and the cyclohexadienone alkyne is the determining factor controlling the enantioselectivity.