Rice (Oryza sativa) is a tropical cereal crop that is severely affected by chilling stress at the seedling stage, although glutinous rice 89-1 (Gr89-1) in Chongqing, China, shows tolerance to low temperatures and overwintering ability. However, little research has been conducted on the mechanisms regulating chilling stress in Gr89-1. In this study, a comprehensive of transcriptional profiles of Gr89-1 seedlings at the three-leaf stage was conducted after a 4 °C treatment for 2, 6, 12, 24, or 48 h. Overall, 2993 differentially expressed genes were detected in Gr89-1 seedlings upon cold exposure. Gene Ontology testing and pathway analysis revealed differentially expressed genes involved in transcriptional regulation, carbohydrate metabolism, plant hormone signal, and cell wall composition. A total of 243 transcription factors were differentially expressed during the cold treatment; in particular, the AP2/EREBP, bHLH, NAC, WRKY, C2H2, and TIFY families were generally upregulated after cold treatment, whereas the mTERF and GNAT families were downregulated. Chilling stress changed the starch and sucrose metabolism, coupled with the accumulation of sucrose and trehalose level, and increases in jasmonic acid level in Gr89-1 seedlings. Furthermore, a number of the cell wall-related genes identified in the present study were also differentially expressed during the cold treatment. The genes and pathways identified in the current study increase our understanding of the mechanisms underlying cold resistance in rice seedlings.