The GADD45 family of proteins consists of three small proteins, GADD45A, GADD45B, and GADD45G, implicated in modulating the cellular response to genotoxic/physiological stressors. Despite similarities in sequence, structure and function, each gadd45 gene is induced differentially by different stress stimuli. Studies on stress-mediated induction of the gadd45 genes have predominantly focused on gadd45a, with knowledge of gadd45b and gadd45g regulation lacking. To generate a more complete understanding of the regulation of gadd45 genes, a comprehensive analysis of stress-mediated induction of human gadd45b has been carried out using human RKO colorectal carcinoma cells as a model system. Novel data indicate that gadd45b induction in RKO cells is regulated by distinct mechanisms in a stress-specific manner. Methylmethane sulfonate (MMS), a DNA alkylating agent, induces gadd45b transcription through a cohort of both constitutive and inducible bound factors, including NFY, Sp1 and Egr1. In contrast, in a hyperosmotic environment generated with sorbitol, gadd45b mRNA is induced exclusively by mRNA stabilization. These findings indicate that the stress-mediated induction of gadd45b is largely distinct from gadd45a. Furthermore, data obtained provide a novel paradigm for stress-response gene induction, indicating that gadd45b induction by distinct stressors, in the same cell type and under the same experimental settings, is differentially regulated at the level of mRNA transcription or mRNA stability. Importantly, this study also provides the groundwork to further examine the regulation of gadd45b expression in in vivo settings using animal models and tissues obtained from normal individuals and cancer patients prior to and after chemotherapeutic intervention.