Chiral compounds usually behave enantioselectively in phyto-biochemical processes. With the increasing application of chiral herbicides, their enantioselective phytotoxicity to plants merits further study, and little information is available in this area. The purpose of this study was to examine the enantioselective phytotoxicity of the herbicide imazethapyr (IM) on the roots of maize (Zea mays L.) seedlings. Enantiomers of IM were separated by HPLC, and their absolute configurations were confirmed as S-(+)-IM and R-(-)-IM by the octant rule. Plant growth measurements and morphological, microscopic, and ultrastructural observations were conducted after treatment with individual IM enantiomers and the racemate. Observations of root morphology showed that the root diameter significantly increased, whereas the root volume, surface area, and number of root tips decreased significantly. IM enantiomers selectively damaged root hair growth and significantly reduced the sloughing of border cells from the tips. IM also had adverse effects on cell organelles, such as statocytes, mitochondria, dictyosomes, and endoplasmic reticulum in maize roots. Moreover, cell membranes and cell walls were thicker than usual after IM treatment. All of the results showed the same trend that the R-(-)-IM affected the root growth of maize seedlings more severely than the S-(+)-IM. The inhibition abilities of (+/-)-IM was between S-(+)- and R-(-)-IM. The behavior of the active enantiomer, instead of just the racemate, may have more relevance to the herbicidal effects and ecological safety of IM. Therefore, enantiomeric differences should be considered when evaluating the bioavailability of the herbicide IM.