Heading date is a critical trait for adaptation of rice to different cultivation areas and cropping seasons. We evaluated the heading dates of 1,123 chromosome segments substitution lines (CSSLs) in the genetic background of an elite rice variety Huajingxian74 (HJX74). A CSSL with the substituted segments from Zihui100 exhibited late heading under both natural long-day (NLD) and natural short-day (NSD) conditions, and the late heading phenotype was controlled by two novel epistatic loci on chromosome 8 and chromosome 3, respectively, termed LH8 and EH3. The function of EH3 was dependent on the LH8 genotype through epistatic interaction between EH3(Zihui100) and LH8(Zihui100) alleles. Genetic and molecular characterization revealed LH8 encodes a CCAAT-box-binding transcription factor with Heading date1 (Hd1)-binding activity and may delay flowering by repressing the expression of Early heading date1 (Ehd1). Our work provides a solid foundation for further study on gene interaction in heading date and has application in breeding rice with greater adaptability.