Two-line breeding of wheat thermosensitive cytoplasmic male sterility (TCMS) is the most important and promising approach in wheat heterosis utilization. However, few studies have reported on the abortion mechanism of wheat TCMS lines and the function of genes. In this study, the wheat TCMS line YS3038 was used as the material to reveal the abortion mechanism of pollen. The transcriptome sequencing combined with VIGS technologies was used to identify and validate the function of candidate genes associated with male sterility. Transcriptomics demonstrated that genes in energy metabolism pathways such as starch and sucrose metabolism, oxidative phosphorylation, and glycolysis and tricarboxylic acid cycle were increasingly differentially expressed under different fertility conditions from the early uninucleate stage to the binucleate stage of microspores development that caused abnormal ATP production, lead to male sterility of YS3038. Morphological and cytological observations have shown abnormal degradation of tapetum cells under low temperature conditions, resulting in insufficient nutrient and the energy supply, late development of microspores, and infertility. VIGS of two candidate genes associated with male sterility showed that silencing of the leucine-rich repeat sequence receptor-like protein kinase (TaeRPK) and the fatty acid acyl-CoA reductase 5 (TaeFar5) significantly reduced the seed setting rate of wheat, indicating that TaeRPK and TaeFar5 genes were probably involved in the male fertility conversion of YS3038. This study reveals that energy metabolism plays an important regulatory role in TCMS male fertility, which provides solid theoretical for the production practice of hybrid wheat.