Abnormalities in the signal transducer and activator of transcription (STAT) pathway are involved in the oncogenesis of several cancers. However, the mechanism by which dysregulated STAT5 signaling contributes to the progression of human colorectal cancer (CRC) has not been elucidated. To investigate the role of STAT5 in CRC progression, we depleted STAT5 with a small interfering RNA (siRNA). Our results demonstrate that STAT5 is involved in CRC cell growth, cell cycle progression, invasion and migration through regulation of gene expression, such as Bcl-2, p16(ink4a), p21(waf1/cip1), p27(kip1), E-cadherin, the focal adhesion kinase (FAK), vascular endothelial growth factor (VEGF) and matrix metalloproteinases. In addition, immunohistochemical staining reveals upregulation of STAT5 during CRC tumorigenesis. Moreover, phospho-STAT5 (pSTAT5) is predominantly localized in the cytoplasm of adenomas cells and colon adenocarcinoma cells, but primarily presented in the nucleus of normal colonic epithelium cells. Thus, pSTAT5 protein is shuttled from the nucleus to the cytoplasm in the oncogenesis of CRC, suggesting that activated STAT5 may also have cytoplasmic functions. In support of this hypothesis, we found that STAT5 formed a complex with p44/42 MAPK and SAPK/JNK in CRC cells, suggesting cross talk between STAT5 signaling and the MAPK pathway in the development of human CRC. Our findings illustrate the biological significance of STAT5 signaling in CRC progression, and provide novel evidence that intervention in STAT5 signaling may have potential therapeutic value in the prevention of human colorectal cancer.