The timing of flowering, which is of crucial importance for plant growth and survival, is controlled by intricate pathways. To identify heading date-QTL, we constructed high resolution map on chromosome 3 using heterogeneous inbred family-near isogenic lines (H-NILs) derived from F7 RILs generated by the cross of early-heading japonica rice ‘H143’ and middle-late-heading indica-japonica hybrid cultivar ‘Milyang23’. QTL and subsequent sequence analysis using H-NILs revealed that the gene underlining QTL EH3, which is detected in the region of Hd16, is EL1 encoding casein kinase I (CKI). Two types of single amino acid substitutions in Ser/Thr kinase domain of EL1 were found in various cultivars, among which H-NIL(eh3) caused loss of function in EL1 demonstrated by altered heading date and GA response. Moreover, the phosphorylation of EL1 appears to involve in EL1 activity to regulate heading date. Transcriptional analysis clearly indicated that H-NIL(EH3) suppresses heading under LD conditions by down-regulating Ehd1, there by Hd3a and RFT1 expressions were not induced, suggesting that EL1 is photoperiod-sensitive and functions as a LD-specific suppressor of heading. Further characterization suggested that EL1 is likely to involve in anther development and seed settings by regulating GAMYB expression. Our study demonstrated that the genetic basis of natural variation occurred in ‘H143’ was revealed by QTL analysis using H-NILs, and EH3/EL1 function is crucial for heading and development in rice. The genetic natural variation of H-NIL(eh3) may have contributed to adaptation of rice cultivation to the higher regions by regulating the expression of rice flowering activator genes and GA signaling.