The intestinal barrier constitutes the largest surface of the human body communicating with the external environment. Alterations affecting elements of intestinal wall may lead to increased intestinal permeability and resulting translocation of bacteria or its components to the bloodstream in the form of the "leaky gut syndrome" (LGS). One of the most common causes of LGS is the disruption of tight junctions (TJ) maintained by tight junction proteins (TJP). LGS and associated alterations in TJP are observed in numerous gastrointestinal (GI) diseases, including inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC). Current literature indicates the key role of LGS in many pathological processes, further emphasizing the need for effective pharmacological approaches to treat this syndrome. One of the potential pharmacological targets in LGS treatment are members of the cytochrome P450 (CYP450) superfamily. By affecting intestinal permeability, they may lead to LGS development. It was found that the expression of CYP8B1 synthesizing cholic acid and CYP26 degrading all-trans retinoic acid indirectly influence TJs. CYP2E1 responsible for the metabolism of a wide variety of chemicals, including ethanol, plays a crucial role in the impairment of the intestinal wall. Contrarily, the overexpression of CYP27B1 has a protective effect on the intestinal integrity. CYP1A1, CYP2A6, CYP2J2 and CYP3A were also suggested to influence the GI tract, through their capability to metabolize serotonin, nicotine, endocannabinoids and gemcitabine, respectively. This review summarizes the findings on the role of CYP450 isoforms in intestinal hyperpermeability and their potential involvement in the pathophysiology of LGS.