Bacteria that exhibit ionic-liquid (IL) tolerance are useful in chemical industries using renewable carbon sources pretreated by ILs to produce biofuels and fine chemicals. Although an IL, 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), has a remarkable ability to solubilize wood components, [BMIM]Cl inhibits the growth of various bacterial hosts useful for bioconversion. We previously isolated a 10% [BMIM]Cl-tolerant bacterium Bacillus amyloliquefaciens CMW1. Here we report novel mechanisms of tolerance to [BMIM]Cl in strain CMW1 and a novel major facilitator superfamily (MFS) transporter coded by an ionic-liquid tolerance (ILT) gene. First, using CMW1 cells grown in the presence or absence of 10% [BMIM]Cl, whole-transcriptome analysis and differentially expressed gene analysis were performed. Probable mechanisms of tolerance to [BMIM]Cl include uptake of osmoprotectants from the culture medium toward CMW1 cells and the export of [BMIM] cations that accumulated in CMW1 cells. The finding represents a first step in elucidation of the mechanisms of IL resistance in Gram-positive bacteria. Second, we conferred tolerance to 5% [BMIM]Cl on [BMIM]Cl-susceptible Brevibacillus choshinensis using ILT gene. This finding provides a notable basis for engineering IL-tolerant bacterial hosts that are applicable for the effective and sustainable production of industrially important chemicals.