Longan (Dimocarpus longan) is an important subtropical evergreen fruit tree (family Sapindaceae) in southern China and exhibits a high commercial and nutritional value. Endocarp browning of longan fruit once harvested considerably reduces the commercial value. The excessive accumulation of reactive oxygen species (ROS) can induce the rapid endocarp browning of longan fruit during storage, but the ROS‐mediated mechanism remains relatively unclear. In this investigation, a hydrogen peroxide scavenger, 1,3‐dimethylthiourea (DMTU), was used to treat longan fruit to elucidate the physiological and molecular bases of the ROS‐mediated endocarp browning of longan fruit. Application of 5 mM DMTU significantly delayed endocarp browning, inhibited respiration rate, suppressed the accumulations of H2O2 and malondialdehyde (MDA), and activated the antioxidant system of longan fruit during storage, such as 3.8 of the DMTU‐treated fruit versus 1.6 of non‐DMTU‐treated fruit of the browning index, 398 versus 346 μg kg−1 S−2 of the respiration rate, and 47.5 versus 37.8 nmol g−1 of the MDA content after 6 days of storage. Furthermore, the DMTU treatment significantly affected the expression levels of the H2O2 receptors and hydrogen peroxide‐induced Ca2+ increases (HPCA)‐LIKEs (HPCALs). Additionally, the regulatory network comprising transcription factors controlling the HPCAL expression in longan was constructed. The major transcription factors, including GATA1, GATA28, TCP20, and AGL20, may contribute to the ROS‐mediated endocarp browning of longan fruit during storage by regulating the HPCAL expression levels. This study could provide new insights into the involvement of the HPCALs in the ROS‐mediated endocarp browning of longan fruit during storage.