The ubiquitously expressed small GTPase RhoA is essential for embryonic development and mutated in different cancers. Functionally, it is well described as a regulator of the actin cytoskeleton, but its role in gene regulation is less understood. Using primary mouse keratinocytes with a deletion of the RhoA gene, we have now been exploring how the loss of RhoA affects gene expression. Performing transcription factor reporter assays, we found a significantly decreased activity of a RAR luciferase reporter in RhoA-null keratinocytes. Inhibition of the RhoA effector ROCK in control cells reproduced this phenotype. ATRA and retinal, but not retinol increased RAR reporter activity of keratinocytes with impaired RhoA/ROCK signalling, suggesting that retinol metabolism is regulated by RhoA/ROCK signalling. Furthermore a significant percentage of known ATRA target genes displayed altered expression in RhoA-null keratinocytes. These data reveal an unexpected link between the cytoskeletal regulator RhoA and retinoid signalling and uncover a novel pathway by which RhoA regulates gene expression.