Crystal leaf mutants of Cymbidium ensifolium have high ornamental value due to their leaves being white transparent shape, but their formation mechanism is not clear. Taking C. ensifolium ‘Tiegushuijing’ as the material, this study intends to explore the reasons for the formation of crystal leaf mutants of C. ensifolium by observation of microstructure and ultrastructure and RNAseq. The results showed that： (1) the crystal leaves of C. ensifolium ‘Tiegushuijing’ were thinner than the green leaves, the number of mesophyll cells was significantly reduced, the cell shape was irregular, and the chloroplast content was small, a significant reduction in the number of stomata in the epidermis of the crystal leaf than the green leaves; the chloroplast structure of mesophyll cells in crystal leaves was poorly developed, the chloroplast double membrane and thylakoid membrane were blurred, and there were a large number of osmiophilic granule in the cells. (2) The RNAseq data results showed that the expression levels of genes related to photosynthesisantennae and photosynthesis metabolic pathways were decreased in crystal leaves, while the expression levels of genes related to pigment metabolic pathways were increased. We speculated that the reason for the formation of crystal leaves of C. ensifolium is that the expression of genes related to photosynthesis are reduced, the chloroplast is poorly developed, and the chlorophyll synthesis is blocked, thereby forming white transparent leaves.