ATOH1 is a basic Helix-Loop-Helix transcription factor crucial for hair cell (HC) differentiation in the inner ear. In order to identify ATOH1 target genes, we performed a genome-wide expression profiling analysis in cells expressing ATOH1 under the control of a tetracycline-off system and found that HES6 expression is induced by ATOH1. We performed in situ hybridisation and showed that the rise and fall of Hes6 expression closely follow that of Atoh1 in cochlear HC. Moreover, electrophoretic mobility shift assays and luciferase assays show that ATOH1 activates HES6 transcription through binding to three clustered E boxes of its promoter.