β-defensins are small cationic antimicrobial peptides secreted by diverse cell types including colonic epithelial cells. Human β-defensins form an essential component of the intestinal lumen in innate immunity. The defensive mechanisms of β-defensins include binding to negatively charged microbial membranes that cause cell death and chemoattraction of immune cells. The antimicrobial activity of β-defensin is well reported in vitro against several enteric pathogens and in non-infectious processes such as inflammatory bowel diseases, which alters β-defensin production. However, the role of β-defensin in vivo in its interaction with other immune components in host defense against bacteria, viruses and parasites with more complex membranes is still not well known. This review focuses on the latest findings regarding the role of β-defensin in relevant human infectious and non-infectious diseases of the colonic mucosa. In addition, we summarize the most significant aspects of β-defensin and its antimicrobial role in a variety of disease processes.