Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2–3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)—COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.