The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.