The mechanisms regulating flowering have been extensively studied and the roles of many environmental signals in this process have been reported. However, little is known on the relationship between iron deficiency and flowering regulation, although the response mechanism to iron deficiency has been studied for decades. In this study, we observed that the flowering time of wild-type Arabidopsis thaliana was significantly repressed by iron deficiency under long days. Phenotype analysis showed that iron deficiency delayed flowering of Arabidopsis through the iron deficiency-induced transcription factors bHLH38, bHLH100, and bHLH101 (bHLH38/100/101), which redundantly regulated flowering time and expression of FLOWERING LOCUS T (FT) specifically under long days. Genetic analysis indicated that disruption of FT expression suppressed the early-flowering phenotype of bhlh38/100/101 triple-mutant plants, indicating that bHLH38/100/101 are dependent on functional FT. Furthermore, bHLH38/100/101 interacted with CONSTANS (CO), thereby interfering with the transcriptional activation of CO to regulate FT expression. Therefore, the results indicated that iron deficiency affects flowering of Arabidopsis under long days through bHLH38/100/101 & ndash;CO & ndash;FT signaling.