Under autotrophic conditions, highly productive biodiesel production was achieved using a semi-continuous culture system in Neochloris oleoabundans. In particular, the flue gas generated by combustion of liquefied natural gas and natural solar radiation were used for cost-effective microalgal culture system. In semi-continuous culture, the greater part (~80%) of the culture volume containing vegetative cells grown under nitrogen-replete conditions in a first photobioreactor (PBR) was directly transferred to a second PBR and cultured sequentially under nitrogen-deplete conditions for accelerating oil accumulation. As a result, in semi-continuous culture, the productivities of biomass and biodiesel in the cells were increased by 58% (growth phase) and 51% (induction phase) compared to the cells in batch culture, respectively. The semi-continuous culture system using two stage photobioreactors is a very efficient strategy to further improve biodiesel production from microalgae under photoautotrophic conditions.