Mot3 and Rox1 are transcriptional repressors of hypoxic genes. Both factors recently have been found to be involved in the adaptive response to hyperosmotic stress, with an important function in the adjustment of ergosterol biosynthesis. Here, we determine the gene expression profile of a mot3 rox1 double mutant under acute osmostress at the genomic scale in order to identify the target genes affected by both transcription factors upon stress. Unexpectedly, we find a specific subgroup of osmostress-inducible genes to be under positive control of Mot3. These Mot3-activated stress genes also depend on the general stress activators Msn2 and Msn4. We confirm that both Mot3 and Msn4 bind directly to some promoter regions of this gene group. Furthermore, osmostress-induced binding of the Msn2 and Msn4 factors to these target promoters is severely affected by the loss of Mot3 function. The genes repressed by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane. Cell conjugation was the most significantly enriched biological process which was negatively regulated by both factors and by osmotic stress. The mating response was repressed by salt stress dependent on Mot3 and Rox1 function. Taking our findings together, the Mot3 transcriptional regulator has unanticipated diverse functions in the cellular adjustment to osmotic stress, including transcriptional activation and modulation of mating efficiency.