The important plant growth regulator 5-aminolevulinic acid (ALA) could promote low-temperature stress tolerance of many plants; however, the underlying mechanisms remain to be elucidated. We investigated the effects of exogenously applied ALA on seedling morphology, antioxidant enzyme activity and photosynthetic capacity of maize (Zea mays L.) seedlings under low-temperature stress. Two cultivars, low-temperature-sensitive cv. Suiyu 13 (SY13) and low-temperature-tolerant cv. Zhengdan 958 (ZD958), were subjected to four treatments: low-temperature without ALA treatment, low-temperature after ALA treatment, normal temperature without ALA treatment, and normal temperature after ALA treatment. Plant morphological growth, proline content, antioxidant enzyme activity and photosynthetic capacity were determined. ALA treatment significantly decreased the inhibitory effects of low-temperature stress on seedling dry weight and increased proline accumulation under low temperatures in ZD958. Pre-application of ALA significantly improved superoxide dismutase and catalase activities in SY13 under low-temperature stress. Furthermore, treating maize seedlings with ALA resulted in significant enhancement of ribulose-1,5-bisphosphate (RuBP) carboxylase activity under low-temperature stress in both cultivars. Pre-treatment with ALA relieved the damage caused by low-temperature stress to maize seedlings, particularly in the low-temperature-sensitive cultivar. Therefore, ALA at appropriate concentrations may be used to prevent reductions in maize crop yield due to low-temperature stress.