Overactivation of apoptosis signal-regulating kinase 1 (ASK1) is associated with inflammatory responses, reactive oxygen species overproduction, cell death, and fibrosis in many tissues. Development of ASK1 activity inhibitors, such as ATP-competitive kinase inhibitors, has been tested for treatments of metabolic dysfunction-associated steatohepatitis (MASH) and diabetes, though great challenges has been met in efficacy and undesirable side effects. We aim to develop a compound that allosterically targets ASK1 activation to inhibit inflammation more effectively. By a screen with a combination of computational and experimental approaches, we identify a potent allosteric disruptor of ASK1 homodimerization, named H43. H43 has strong anti-inflammatory effect accompanied by suppression of the mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase (JNK) signaling in cells. In vivo, H43 significantly enhances survival rates, reduces inflammatory mediators, and attenuates lung damage in endotoxic male mice. H43 also mitigates MASH by inhibiting inflammation, alleviating liver damage and fibrosis in male mice. This study identifies a chemical tool to allosterically modulate ASK1 activity and a new approach for therapeutic potential of controlling inflammation and fibrosis.