Trehalose has been shown to protect bacterial cells from environmental stress. Its uptake and osmoprotective effect in Clostridium perfringens were investigated by comparing wild type C. perfringens ATCC 13124 with a fluoroquinolone- (gatifloxacin-) resistant mutant. In a chemically defined medium, trehalose and sucrose supported the growth of the wild type but not that of the mutant. Microarray data and qRT-PCR showed that putative genes for the phosphorylation and transport of sucrose and trehalose (via phosphoenolpyruvate-dependent phosphotransferase systems, PTS) and some regulatory genes were downregulated in the mutant. The wild type had greater tolerance than the mutant to salts and low pH; trehalose and sucrose further enhanced the osmotolerance of the wild type to NaCl. Expression of the trehalose-specific PTS was lower in the fluoroquinolone-resistant mutant. Protection of C. perfringens from environmental stress could therefore be correlated with the ability to take up trehalose.