An analytical approach was developed by coupling QuEChERS extraction with UHPLC-MS/MS to separate and quantify four stereoisomers of isocycloseram in cabbage and soil. The stereoisomers, including cis-(+)-(5R,4S) ISR, cis-(-)-(5S,4R) ISM, trans-(+)-(5R,4R) ISO, and trans-(-)-(5S,4S) ISS, were baseline-resolved on a Chiralcel OJ-3R column using acetonitrile/water (60:40, 0.3% formic acid). The method showed good linearity (R2 > 0.998), LOQs of 0.01 mg kg-1, and recoveries of 85.33-109.51% (RSDs ≤11.64%). Field trials revealed that cis-isomers, particularly ISM, dissipated faster than trans-isomers (ISO, ISS), with half-lives of 2.11-4.95 days. DFT calculations indicated a lower HOMO-LUMO gap and reduced stability for ISM, with ISS also showing lower stability. Washing with 1% citric acid removed up to 88.35% of ISM and over 76% of the other isomers, correlating with molecular polarity and electrostatic potential. This study provides a tool for chiral pesticide analysis and insights into stereoselective behavior, aiding environmental fate and dietary risk assessment.