Abstract: Petroleum contamination in terrestrial environments caused by industrial activities is a significant problem that has received considerable attention. Carbon and nitrogen isotopic compositions (δ¹³C and δ¹⁵N) effectively describe the behavior of plants and soils under petroleum contamination stress. To better understand plant and soil responses to petroleum-contaminated soil, δ¹³C and δ¹⁵N values of the plants (Trifolium repens, Leguminosae with C₃ photosynthesis pathway, and Agropyron cristatum with C₄ photosynthesis pathway) and the soil samples under one-month exposure to different extents of petroleum contamination were measured. The results showed that petroleum contamination in the soil induced the soil δ¹⁵N values to increase and δ¹³C values to decrease; from 1.9‰ to 3.2‰ and from −23.6‰ to −26.8‰, respectively. However, the δ¹³C values of Agropyron cristatum decreased from −29.8‰ to −31.6‰, and the δ¹³C values of Trifolium repens remained relatively stable from −12.6‰ to −13.1‰, indicating that they have different coping strategies under petroleum-contaminated soil conditions. Moreover, the δ¹⁵N values of Trifolium repens decreased from 5.6‰ to 0.8‰ near the air δ¹⁵N values under petroleum-contaminated soil, which implies that their nitrogen fixation system works to reduce soil petroleum stress. The δ¹³C and δ¹⁵N values of Agropyron cristatum and Trifolium repens reflect changes in the metabolic system when they confront stressful environments. Therefore, stable isotopic compositions are useful proxies for monitoring petroleum-contaminated soil and evaluating the response of plants to petroleum contamination stress.