Short-chain fatty acids (SCFAs) are a group of organic compounds produced by the fermentation of dietary fibre by the human gut microbiota. They play diverse roles in different physiological processes of the host with implications for human health and disease. This Review provides an overview of the complex microbial metabolism underlying SCFA formation, considering microbial interactions and modulating factors of the gut environment. We explore the multifaceted mechanistic interactions between SCFAs and the host, with a particular focus on the local actions of SCFAs in the gut and their complex interactions with the immune system. We also discuss how these actions influence intestinal and extraintestinal diseases and emerging therapeutic strategies using SCFAs.