Pollination is a crucial stage in plant reproductive process. The self-compatibility (SC) and self-incompatibility (SI) mechanisms determined the plant genetic diversity and species survival. D. chrysanthum is a highly valued ornamental and traditional herbal orchid in Asia but has been declared endangered. The sexual reproduction in D. chrysanthum relies on the compatibility of pollination. To provide a better understanding of the mechanism of pollination, the differentially expressed proteins (DEP) between the self-pollination (SP) and cross-pollination (CP) pistil of D. chrysanthum were investigated using proteomic approaches—two-dimensional electrophoresis (2-DE) coupled with tandem mass spectrometry technique. A total of 54 DEP spots were identified in the two-dimensional electrophoresis (2-DE) maps between the SP and CP. Gene ontology analysis revealed an array of proteins belonging to following different functional categories: metabolic process (8.94%), response to stimulus (5.69%), biosynthetic process (4.07%), protein folding (3.25%) and transport (3.25%). Identification of these DEPs at the early response stage of pollination will hopefully provide new insights in the mechanism of pollination response and help for the conservation of the orchid species.