Rehmannia glutinosa L. is a perennial herbaceous species belonging to the Scrophulariaceae family. Its tuberous roots are used in Chinese medicine, but its productivity tends to fall drastically after its first cropping season. While the physiological and environmental factors responsible for this yield decline are to an extent understood, the changes in gene expression associated with continuous cropping have not as yet been documented. We have attempted to define which genes are differentially expressed in the first year and second year planting materials by creating both a forward and a reverse subtractive library, containing, respectively, 232 and 214 cDNA fragments, of which 200 and 195 shared homology with known function sequences found in other plant species. The resulting functional assignment of 16 of these sequences, along with their patterns of spatial and temporal expression, suggested that continuous cropping disturbed the initiation of Ca2+ signal transduction and ethylene synthesis, and repressed DNA replication, RNA transcription and protein synthesis. Irrigation of Ca2+ signal blocker in roots of plants could alleviate the syndromes of continuously cropping disease in R. glutinosa. The molecular regulatory network underlying the continuous cropping disease has been sketched out, and this should serve as a basis for revealing the genetic causes of the syndrome in R. glutinosa.