Abstract: Due to massive exploitation and use of fossil fuel such as petroleum, coal and natural gas, atmospheric CO₂ concentration has been increasing, which not only accelerated global warming, but also affected the survival and distribution of animals and plants on the earth with far-reaching impacts on the ecosystem. This research was carried out to explore the effects of elevated CO₂ concentration and pest population density of Acyrthosiphon pisum on chemical substances in the leaves of Medicago sativa. The objective was assessed the effects of elevated CO₂ concentration and pea aphid density on the physiology and biochemistry of M. sativa. The nutrients and secondary metabolites in M. sativa leaves were determined by cultivating M. sativa seedlings attacked by 10-day old pea aphids of 10 head·plant^-1, 20 head·plant^-1, 30 head·plant^-1 and 0 head·plant^-1 (CK) for one week under three CO₂ concentrations380 μL·L^-1 (CK), 550 μL·L^-1 and 750 μL·L^-1 in CO₂ gradient chamber. The results indicated that the contents of soluble protein, soluble carbohydrate and starch increased after aphid sucking of 30 head·plant^-1 with increasing CO₂ concentration. At 750 μL·L^-1 CO₂ concentration, they were respectively 11.62 times, 0.49 times and 0.24 times higher than those under CK, respectively. Also the contents of flavone, total polyphenols and simple phenols increased significantly. Furthermore, the contents of starch and simple phenols increased and then decreased with increasing degree of damage under the same CO₂ concentration. Comparatively, contents of soluble carbohydrate, total polyphenols and tannin were significant differences between aphid densities of 30 head·plant^-1 and 0 head·plant^-1, which increased by 1.66 times, 1.49 mg·g^-1 and 1.09 mg·g^-1 (P < 0.05) compared with those of CK under the highest level of CO₂ concentration. The results indicated that nitrogen fixing legume plants were more likely to adapt to increased CO₂ concentration and thereby enhanced self-induced resistance to insect pests.