Dicer and Dicer-like (DCL) proteins are key components in small RNA biogenesis. DCLs form a small protein family in plants whose diversification time dates to the emergence of mosses (Physcomitrella patens). DCLs are ubiquitously but not evenly expressed in tissues, at different developmental stages, and in response to environmental stresses. In Arabidopsis, AtDCL1, AtDCL2, and AtDCL4 exhibit similar expression pattern during the leaf or stem development, which is distinguished from AtDCL3. However, distinct expression profiles for all DCLs are found during the development of reproductive organs flower and seed. The grape VvDCL1 and VvDCL3 may act sequentially to face the fungi challenge. Overall, the responses of DCLs to drought, cold, and salt are quite different, indicating that plants might have specialized regulatory mechanism in response to different abiotic stresses. Further analysis of the promoter regions reveals a few of cis-elements that are hormone- and stress-responsive and developmental-related. However, gain and loss of cis-elements are frequent during evolution, and not only paralogous but also orthologous DCLs have dissimilar cis-element organization. In addition to cis-elements, AtDCL1 is probably regulated by both ath-miR162 and ath-miR414. Posterior analysis has identified some critical amino acid sites that are responsible for functional divergence between DCL family members. These findings provide new insights into understanding DCL protein functions.