Diastereomer- and enantiomer-specific accumulation and biotransformation of hexabromocyclododecane (HBCD) in maize (Zea mays L.) were investigated. Molecular interactions of HBCD with plant enzymes were further characterized by homology modeling combined with molecular docking. The (-)α-, (-)β-, and (+)γ-HBCD enantiomers accumulated to levels in maize significantly higher than those of their corresponding enantiomers. Bioisomerization from (+)/(-)-β- and γ-HBCDs to (-)α-HBCD was frequently observed, and (-)γ-HBCD was most easily converted, with bioisomerization efficiency of 90.5 ± 8.2%. Mono- and dihydroxyl HBCDs, debrominated metabolites including pentabromocyclododecene (PBCDe) and tetrabromocyclododecene (TBCDe), and HBCD-GSH adducts were detected in maize roots. Patterns of hydroxylated and debrominated metabolites were significantly different among HBCD diastereomers and enantiomers. Three pairs of HBCD enantiomers were selectively bound into the active sites and interacted with specific residues of maize enzymes CYP71C3v2 and GST31. (+)α-, (-)β-, and (-)γ-HBCDs preferentially bound to CYP71C3v2, whereas (-)α-, (-)β-, and (+)γ-HBCDs had strong affinities to GST31, consistent with experimental observations that (+)α-, (-)β-, and (-)γ-HBCDs were more easily hydroxylated, and (-)α-, (-)β-, and (+)γ-HBCDs were more easily isomerized and debrominated in maize compared to their corresponding enantiomers. This study for the first time provided both experimental and theoretical evidence for stereospecific behaviors of HBCD in plants.