Brassinosteroids (BRs) play critical roles in a wide range of plant developmental processes. However, it is unknown whether and how BRs mediate the effect of high temperature (HT) stress during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile (PTSGMS) rice (Oryza sativa L.) lines. This study investigated the question. Three pot-grown PTSGMS rice lines were subjected to HT stress during anthesis. The contents of 24-epibrassinolide (24-EBL) and 28-homobrassinolide (28-HBL), the major forms of BR in rice plants, and levels of reactive oxygen species (ROS) or antioxidants (AOS), hydrogen peroxide (H2O2), 1-aminocylopropane-1-carboxylic acid (ACC), ascorbic acid (AsA), and catalase activity in pistils, were determined. HT stress significantly reduced the contents of both 24-EBL and 28-EBL relative to those under normal temperatures, but the reduction varied by PTSGMS line. A line with higher BR contents under HT stress showed lower contents of ACC and H2O2, higher catalase activity and AsA content in pistils, and higher fertilization rate, seed-setting rate, and seed yield when the line was crossed with a restorer line, indicating that higher levels of BRs increase HT stress resistance. Applying 24-EBL, 28-HBL or an inhibitor of BR biosynthesis confirmed the roles of BRs in response to HT stress. The results suggest that BRs mediate the effect of HT stress on pistil activity during anthesis and alleviate the harm of HT stress by increasing AOS and suppressing ROS generation.