Unravelling the features of the whole-body response to acute stress exposures is critical to understand this neglected building block of chronic stress. A single acute stress exposure rapidly modulates gut-brain axis signalling including intestinal permeability, but the mechanisms are unclear. Microbially-produced metabolites such as short-chain fatty acids (SCFA) are key effectors within the gut-brain axis which can affect gut and brain function. The aim of this work is to determine if acute stress regulates SCFA production in the gut and to understand the associated implications for gastrointestinal and brain barrier function. Stress reduced caecal SCFA concentrations, primarily butyrate and acetate. These SCFAs prevented LPS-induced disruption of gut and brain barrier function in a dose-dependent manner in in vitro models. This functional protection was associated with altered tight-junction abundance and morphology. These results provide a better understanding of the role SCFAs have on barriers following acute stress.