Flowering time is one of the most important agronomic traits in wheat production. A proper flowering time might contribute to the reduction or avoidance of biotic and abiotic stresses, adjust plant architecture, and affect the yield and quality of grain. In this study, TaTOE1-B1 in wheat produced three transcripts (TaTOE1-B1-1, TaTOE1-B1-2, and TaTOE1-B1-3) by alternative splicing. Compared to the longest transcript, TaTOE1-B1-1, TaTOE1-B1-3 has a deletion in the sixth exon (1219–1264 bp). Under long-day conditions, the heterologous overexpression of the TaTOE1-B1-3 gene delayed flowering, prolonged the vegetative growth time, and enlarged the vegetative body of Arabidopsis, but that of TaTOE1-B1-1 did not. As typical AP2 family members, TaTOE1-B1-1 and TaTOE1-B1-3 are mainly located in the nucleus and have transcriptional activation activities; the transcriptional activation region of TaTOE1-B1-3 is located in the C-terminal. In TaTOE1-B1-3 overexpression lines, the expression of flowering-related AtFT and AtSOC1 genes is significantly downregulated. In addition, this study confirms the protein–protein interaction between TaTOE1-B1-3 and TaPIFI, which may play an important role in flowering inhibition. These results provide a theoretical basis for the precise regulation of wheat flowering time.