Recent research highlighted the essential role of gut microbiota in maintaining human health, especially in preventing immune-related disorders [1]. The incidence of allergies and inflammatory and autoimmune diseases has dramatically increased over the past century, which parallels the significant changes in our environment. Factors such as stress, pollutants, a low-fiber diet, infections, and excessive use of antibiotics cause an imbalance in the gut microbiota, leading to persistent dysbiosis. This dysbiosis can then modulate the immune system both locally in the intestine and at the systemic level, resulting in various immune-related diseases [2]. The development of a balanced gut microbiota during the perinatal period and the maintenance of its microbial composition in adulthood are achieved by several factors, including secretory immunoglobulin A (sIgA) from plasma cells, mucins from goblet cells, and antimicrobial peptides (AMPs) from intestinal epithelial cells (IECs) or commensal bacteria themselves [3, 4]. Thanks to a long evolutionary adaptation, AMPs represent a dynamic chemical barrier that actively kills potential invaders and preserves beneficial commensals. AMPs are found in a wide diversity of life forms from microorganisms to humans. They represent a large family of short cationic peptides, with direct microbicide potential toward pathogenic microorganisms, including viruses, fungi, and bacteria. Due to their cationic nature, AMPs bind bacterial membrane and form pores leading to bacterial cell death while not harming the host’s own cells [5]. The defensins and cathelicidins are the most representative classes of AMPs in the intestinal tract being differently expressed by highly specialized Paneth cells or undifferentiated epithelial cells [6]. Beyond their microbicidal properties, AMPs also play important physiological and immunological roles in the intestine and many organs, placing them at the delicate intersection of many biological processes. In this chapter, we review the molecular mechanism of AMP antimicrobial activity in the gut and how it affects the microbiome. We discuss how AMP dysfunction is involved in diseases indirectly through the microbiota and directly in shaping the host immune response outside the gut.