Heterosigmaakashiwo is an algal species that causes harmful algal blooms (HABs) with strong hemolytic toxicity on coastal aquatic organisms. This study investigated the mechanism of growth inhibition and changes in hemolytic toxin contents in algal culture after exposure to hypoxanthine, a compound secreted by algicidal bacterium Bacillus sp.strain B1. An algal inhibition rate of 86% was observed with 1.0 mM hypoxanthine treatment on day 15. The levels of superoxide dismutase and catalase in algal cell culture increased while that of glutathione decreased during the treatment. In addition,the level of hemolytic toxin contents increased on day 3 under hypoxanthine treatment, and significantly decreased on days 6, 9, 12, and 15. Twelve fatty acids in H.akashiwo were detected by GC-MS, and the changes in the contents of C16, C18, C18:4ω3, and C20:5ω3 in the treatment group were consistent with the change in hemolytic toxin content. The four fatty acids were tested for hemolysis and it was observed that the hemolysis rate of 25 μg/mL C18:4ω3 and 5 μg/mL C20:5ω3 reached more than 80%, but C16 and C18 exhibited no hemolytic capability.Therefore, our results showed that hypoxanthine inhibited the growth of H. akashiwo through the changes of levels of antioxidants and hemolytic toxin content in the cultures, and fatty acids C18:4ω3 and C20:5ω3 were contributors to hemolytic toxins. The results confirmed that hypoxanthine is a potential algal inhibitor to prevent HABs.