Conversion between a cyst and trophozoite stage is essential to disease transmission and pathogenesis in the parasitic protist Entamoeba histolytica. A transcriptomic analysis of E. histolytica cysts and trophozoites has recently been accomplished, but the molecular basis of the regulation of encystation is not known. We have now identified a developmentally regulated Myb protein (belonging to the SHAQKY family of Myb proteins), which controls expression of a subset of amoebic stage-specific genes. Overexpression of the nuclear localized Myb protein resulted in a transcriptome that overlapped significantly with the expression profile of amoebic cysts. Analysis of promoters from genes regulated by the Myb protein identified a CCCCCC promoter motif to which amoebic nuclear protein(s) bind in a sequence-specific manner. Chromatin immunoprecipitation demonstrated that the E. histolytica Myb protein binds to promoters of genes which contain the CCCCCC motif and which are regulated by the Myb protein. This work is the first identification of a transcription factor, which regulates expression of a subset of stage-specific genes in E. histolytica. Identification of transcriptional regulatory networks that control developmental pathways will provide novel insights into the biology of this important human pathogen.