Wheat is one of the most important staple crops worldwide. Fusarium head blight severely reduces wheat yield and quality. Cultivation of a novel type of cleistogamous wheat mutant, ZK001, which was created by static magnetic field treatment, is a new strategy for controlling Fusarium head blight. However, little is known about the mechanism of cleistogamy in wheat. The present study demonstrated that anthers of ZK001 were retained on the glumes at all flowering stages, whereas those of YM18 were extruded from the paleae and lemmae. There was a clear difference in the morphological characteristics of lodicules between YM18 and ZK001. Lodicule calcium and potassium contents were significantly higher in YM18 than in ZK001 from white to yellow anther stages. In Fusarium head blight resistance, the diseased kel rate and deoxynivalenol content of ZK001 were markedly lower than those of YM18 and QM725. Comparative transcriptome analysis of YM18 and ZK001 was performed to identify regulatory mechanisms of cleistogamy. The main differentially expressed genes identified in the spikelets of YM18 and ZK001 at the green anther stage were associated with cell walls, carbohydrates, phytohormones, water channel, and ion binding, transport, and homeostasis. These differentially expressed genes may play an important role in regulating cellular homeostasis, osmotic pressure, and lodicule development. The results indicate that ZK001 lost the ability to push the lemmae and paleae apart during the flowering stage because of the thin lodicules. ZK001 was speculated to provide structural barriers for Fusarium head blight during the flowering stage. The thin lodicule of ZK001 results from low levels of soluble sugar, calcium ions, and potassium ions in the lodicules. These levels are regulated by differentially expressed genes.