The NO-sensitive NsrR repressor of Bacillus subtilis, which carries a [4Fe-4S] cluster, controls transcription of nasD and hmp (class I regulation) under anaerobic conditions. Here, we describe another class of NsrR regulation (class II regulation) that controls a more diverse collection of genes. Base substitution analysis showed that [4Fe-4S]-NsrR recognizes a partial dyad symmetry within the class I cis-acting sites, whereas NO-insensitive interaction of NsrR with an A+T-rich class II regulatory site showed relaxed sequence specificity. Genome-wide transcriptome studies identified genes that are under the control of the class II NsrR regulation. The class II NsrR regulon includes genes controlled by both AbrB and Rok repressors, which also recognize A+T-rich sequences, and by the Fur repressor. Transcription of class II genes was elevated in an nsrR mutant during anaerobic fermentative growth with pyruvate. Although NsrR binding to the class II regulatory sites was NO insensitive in vitro, transcription of class II genes was moderately induced by NO, which involved reversal of NsrR-dependent repression, suggesting that class II repression is also NO sensitive. In all NsrR-repressed genes tested, the loss of NsrR repressor activity was not sufficient to induce transcription as induction required the ResD response regulator. The ResD-ResE signal transduction system is essential for activation of genes involved in aerobic and anaerobic respiration. This study indicated coordinated regulation between ResD and NsrR and uncovered a new role of ResD and NsrR in transcriptional regulation during anaerobiosis of B. subtilis.