ERG, a member of the ETS transcription factor family, is frequently overexpressed in prostate cancer as a result of its fusion to the androgen-responsive Tmprss2 gene. Different genomic rearrangements and alternative splicing events around the junction region lead to multiple combination of Tmprss2:ERG fusion transcripts that correlate with different tumor aggressiveness, but their specific functions and biological activities are still unclear. The complexity of ERG expression pattern is compounded by the use of alternative promoters, splice sites, polyadenylation sites and translation initiation sites in both the native and fusion contexts. Our systematic characterization of native ERG and Tmprss2:ERG variants reveals that their different oncogenic potential is impacted by the status of the Ets domain and the configuration of the 5' UTR region. In particular, expression and activity of functional ERG and Tmprss2:ERG variants are influenced both by translation initiation signals within the different isoforms and by inhibitory upstream Open Reading Frames (uORF) in their 5' UTRs. Stable expression of ERG and Tmprss2:ERG variants promoted cell migration/invasion, induced a block of proliferation and induced a senescence-like state, suggesting a role for these variants in the prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products, a group of related native ERG isoforms is also highly over-expressed in fusion-carrying prostate cancers, and share the same translation initiation site (in ERG exon 4) with the commonly observed Tmprss2 exon1 joined to ERG exon 4 (T1:E4) fusion-derived variant. Usage of this ATG can be preferentially down-regulated by directed antisense-based compounds, possibly representing the basis of a targeted approach that distinguishes between tumor-associated and normal ERG.