The fusion between EWS and ETS family members is a key oncogenic event in Ewing tumors and important EWS-FLI1 target genes have been identified. However, until now, the search for EWS-FLI1 targets has been limited to promoter regions and no genome-wide comprehensive analysis of in vivo EWS-FLI1 binding sites has been undertaken. Using a ChIP-Seq approach to investigate EWS-FLI1-bound DNA sequences in two Ewing cell lines, we show that this chimeric transcription factor preferentially binds two types of sequences including consensus ETS motifs and microsatellite sequences. Most bound sites are found outside promoter regions. Microsatellites containing more than 9 GGAA repeats are very significantly enriched in EWS-FLI1 immunoprecipitates. Moreover, in reporter gene experiments, the transcription activation is highly dependent upon the number of repeats that are included in the construct. Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation. Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis.