RNA-binding protein Hfq is a key component of the adaptive responses of many proteobacterial species including Escherichia coli, Salmonella enterica and Vibrio cholera. In these organisms, the importance of Hfq largely stems from its participation to regulatory mechanisms involving small non-coding RNAs. In contrast, the function of Hfq in Gram-positive bacteria has remained elusive and somewhat controversial. In the present study, we have further addressed this point by comparing growth phenotypes and transcription profiles between wild-type and an hfq deletion mutant of the model Gram-positive bacterium, Bacillus subtilis. The absence of Hfq had no significant consequences on growth rates under nearly two thousand metabolic conditions and chemical treatments. The only phenotypic difference was a survival defect of B. subtilis hfq mutant in rich medium in stationary phase. Transcriptomic analysis correlated this phenotype with a change in the levels of nearly one hundred transcripts. Albeit a significant fraction of these RNAs (36%) encoded sporulation-related functions, analyses in a strain unable to sporulate ruled out sporulation per se as the basis of the hfq mutant's stationary phase fitness defect. When expressed in Salmonella, B. subtilis hfq complemented the sharp loss of viability of a degP hfq double mutant, attenuating the chronic σE-activated phenotype of this strain. However, B. subtilis hfq did not complement other regulatory deficiencies resulting from loss of Hfq-dependent small RNA activity in Salmonella indicating a limited functional overlap between Salmonella and B. subtilis Hfqs. Overall, this study confirmed that, despite structural similarities with other Hfq proteins, B. subtilis Hfq does not play a central role in post-transcriptional regulation but might have a more specialized function connected with stationary phase physiology. This would account for the high degree of conservation of Hfq proteins in all 17 B. subtilis strains whose genomes have been sequenced.