Two chickpea (Cicer arietinum L.) lines (AKN 87 and AKN 290) that have different chilling susceptibilities were exposed to 2 chilling temperatures (4 and 2 °C), either cold-acclimated (10 °C) or nonacclimated (25 °C), in order to understand and compare physiological and biochemical changes at the vegetative stage. Chilling temperatures resulted in reduced growth parameters, particularly in cold-acclimated lines, whereas nonacclimated plants exhibited the lowest water contents. Cold acclimation treatment led to protective changes of increased flavonoid, proline, and antioxidant enzyme activities, mostly superoxide dismutase (SOD) and ascorbate peroxidase (APX), in the lines. However, the 10 °C treatment did not significantly influence photosystem II (PSII) activity in chickpea plants. In chilling treatments, cold-acclimated plants exhibited better tolerance; of the 2 lines, AKN 87 had the higher PSII photochemical activity. The chlorophyll contents of lines decreased, while the anthocyanin and flavonoid contents of lines increased at decreasing temperatures. MDA and proline accumulation increased with the severity of the chilling stress. In conclusion, when the plants were exposed to cold acclimation, chilling induced the enhancement of antioxidant enzyme activities, particularly SOD and APX. The results demonstrated that cold acclimation reduced the deteriorative effects of chilling and provided better tolerance, specifically in AKN 87. The line AKN 87 has greater potential for cultivation as a chilling-tolerant cultivar.