Enterotoxigenic produces a myriad of adhesive structures collectively named colonization factors (CFs). CS3 is a CF, which is assembled into fine wiry fibrillae encoded by the gene cluster. In this work we evaluated the influence of environmental cues such as temperature, osmolarity, pH, and carbon source on the expression of CS3 genes. The transcription of major pilin gene was stimulated by growth of the bacteria in colonization factor broth at 37°C; the presence of glycerol enhanced transcription, while glucose at high concentration, high osmolarity, and the depletion of divalent cations such as calcium and magnesium repressed expression. In addition, we studied the role of H-NS, CpxRA, and CRP global regulators in CS3 gene expression. H-NS and CpxRA acted as repressors and CRP as an activator of expression. Under high osmolarity, H-NS, and CpxRA were required for repression. CS3 was required for both, bacterial adherence to epithelial cells and biofilm formation. Our data strengthens the existence of a multi-factorial regulatory network that controls transcription of CS3 genes in which global regulators, under the influence of environmental signals, control the production of this important intestinal colonization factor.