Chitosan is a plasma membrane-perturbing compound consisting of linear chains of beta-1,4-linked glucosamine residues, which at acidic pHs become positively charged. It is extensively used as an antimicrobial compound, yet its mode of action is still unresolved. Chitosan strongly affected the growth of the yeast Saccharomyces cerevisiae, the food spoilage yeast Zygosaccharomyces bailii, and two human-pathogenic yeasts, Candida albicans and Candida glabrata. Microarray analysis of yeast cells treated with sublethal concentrations of chitosan revealed induction of the environmental stress response and three more major transcriptional responses. The first was a rapid and stable Cin5p-mediated response. Cin5p/Yap4p is a transcription factor involved in various stress responses. Deletion of CIN5 led to increased chitosan sensitivity. The second was a Crz1p-mediated response, which is delayed compared to the Cin5p response. Crz1p is a transcription factor of the calcineurin pathway. Cells deleted for CRZ1 or treated with the calcineurin inhibitor FK506 became hypersensitive to chitosan, supporting the notion that the Crz1p-controlled response offers protection against chitosan. The third was a strong Rlm1p-mediated response which ran parallel in time with the Crz1p-regulated response. Rlm1p is a transcription factor of the cell wall integrity pathway, which is activated by cell wall stress. Importantly, chitosan-treated cells became more resistant to beta-1,3-glucanase, which is a well-known response to cell wall stress. We propose that the transcriptional response to chitosan may be representative of other plasma membrane-perturbing compounds.