In this study, 26 indica hybrid rice restorer lines were evaluated for cold tolerance at the seedling stage, and the strongly cold-tolerant restorer line R382 was identified. Using the widely cultivated but relatively cold-sensitive restorer line 9311 as a control, the physiological mechanisms underlying the cold tolerance of R382 at the seedling stage were revealed by comparing differences in cold-related physiological indices and low-temperature-responsive gene expression levels between R382 and 9311. Under identical growth conditions, changes in several physiological parameters of rice seedlings (leaves) of R382 and 9311 were systematically analyzed under both normal temperature and low-temperature stress, including levels of reactive oxygen species (hydrogen peroxide, superoxide anion), malondialdehyde (MDA) content, antioxidant enzyme activities, relative electrolyte leakage, and the osmoprotectant proline content. In addition, real-time quantitative PCR (qRT-PCR) was used to measure the expression levels of genes encoding antioxidant enzymes catalase (OsCATB), iron-superoxide dismutase (OsFe-SOD), ascorbate peroxidase (OsAPX1) and the proline-rich protein gene OsPRP1 under low-temperature stress at the seedling stage. The results showed that after low-temperature treatment, the expression levels of antioxidant enzyme-coding genes in R382 were significantly higher than those in 9311, accompanied by stronger antioxidant enzyme activities, lower accumulation of reactive oxygen species and the toxic compound MDA, and a marked increase in intracellular proline content, along with lower relative electrolyte leakage. These combined physiological and molecular differences contribute to the stronger cold tolerance of R382 at the seedling stage.