The two-component system CBO0366/CBO0365 was recently demonstrated to have a role in cold tolerance of group I Clostridium botulinum ATCC 3502. The mechanisms under its control, ultimately resulting in increased sensitivity to low temperature, are unknown. A transcriptomic analysis with DNA microarrays was performed to identify the differences in global gene expression patterns of the wild-type ATCC 3502 and a derivative mutant with insertionally inactivated cbo0365 at 37 and 15°C. Altogether, 150 or 141 chromosomal coding sequences (CDSs) were found to be differently expressed in the cbo0365 mutant at 37 or 15°C, respectively, and thus considered to be under the direct or indirect transcriptional control of the response regulator CBO0365. Of the differentially expressed CDSs, expression of 141 CDSs was similarly affected at both temperatures investigated, suggesting that the putative CBO0365 regulon was practically not affected by temperature. The regulon involved genes related to acetone-butanol-ethanol (ABE) fermentation, motility, arsenic resistance, and phosphate uptake and transport. Deteriorated growth at 17°C was observed for mutants with disrupted ABE fermentation pathway components (crt, bcd, bdh, and ctfA), arsenic detoxifying machinery components (arsC and arsR), or phosphate uptake mechanism components (phoT), suggesting roles for these mechanisms in cold tolerance of group I C. botulinum. Electrophoretic mobility shift assays showed recombinant CBO0365 to bind to the promoter regions of crt, arsR, and phoT, as well as to the promoter region of its own operon, suggesting direct DNA-binding transcriptional activation or repression as a means for CBO0365 in regulating these operons. The results provide insight to the mechanisms group I C. botulinum utilizes in coping with cold.