The microalga Poterioochromonas malhamensis was found to be capable of accumulating the storage beta-1,3-glucan in soluble form under heterotrophic conditions. In this study, the highest biomass yield of 32.8 g L-1 was achieved by combining the utilization of ammonium chloride as the nitrogen source, simultaneous addition of vitamins B1 and B12 and maintenance of pH at 6.0. Sugar profiling and nuclear magnetic resonance analysis indicated that the P. malhamensis beta-1,3-glucan was composed of glucose with the beta-(1 -> 3) main chain and the beta-(1 -> 6) side chain. Under the optimal cultivation conditions, the cellular beta-1,3-glucan content was up to 55% of the cell dry weight. Moreover, the P. malhamensis beta-1,3-glucan could significantly promote the fin regeneration and improve the in vivo antioxidative activity of zebrafish. This study underpins the feasibility of culturing P. malhamensis under heterotrophic conditions for producing the highly water-soluble bioactive beta-1,3-glucans for food and pharmaceutical applications.