The photo-transformation of dissolved organic matter (DOM), which is ubiquitous in aquatic environments, significantly alters its molecular composition and interactions in surface water environment, affecting its fate in water treatment processes. However, the effect of photo-transformation on the coagulation behavior of DOM remains poorly understood. Herein, the photo-transformation pathways of DOM during simulated sunlight irradiation and its effect on coagulation behavior of DOM were investigated. Reactomics analysis demonstrated the polyphenols in DOM underwent dealkylation reactions, reactions of carboxylic acid, and oxygen addition reactions upon sunlight irradiation, generating molecules with low aromaticity and molecular weight, and low-oxygen-content fragments. Such alterations resulted in a reduction in the relative abundance of the dominant component being C&O-rich lignin-like compounds enriched in –CH2CO2, –2COOH, and –2OH groups, leading to the decrease of DOM removal efficiency. These alterations caused a variation in coagulation mechanisms for DOM removal, shifting from charge neutralization to adsorption bridging after 36 h of simulated sunlight irradiation. This study offers novel insights into the effects of photo-oxidation on the coagulation behaviour of DOM, contributing to the optimization of drinking water treatment and the safe supply of drinking water.