Polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA), have attracted attention owing to their health benefits for humans, as well as their importance in aquaculture and animal husbandry. Establishing a sustainable PUFA supply based on fish oils has been difficult due to their increasing demand. Therefore, alternative sources of PUFAs are required. In this research, we examined the potential of the marine oleaginous diatom Fistulifera solaris as an alternative producer of PUFAs. Optimization of culture conditions was carried out for high cell density cultivation, and a maximal biomass productivity of 1.32 +/- 0.13 g/(L.day) was achieved. By slightly adjusting the culture conditions for EPA production, the maximal EPA productivity reached 135.7 +/- 10.0 mg/(L.day). To the best of our knowledge, this is the highest EPA productivity among microalgae cultured under photoautotrophic conditions. This result indicates that F. solaris is a promising candidate host for sustainable PUFA production.