This study aimed to improve valuable omega-3 fatty acids production in freshwater microalgae at normal temperature by inducing homeoviscous adaptation using CaCl2, which could have a role in decreasing the cellular membrane fluidity followed by increasing the rigidity of cell wall and membranes. At 10 mM CaCl2, simultaneous biomass and lipid production was obtained by Ca2+-based single strategy without considerable sacrifice of cellular logarithmic growth in Chlorella sorokiniana. The cells cultured at 10 mM CaCl2 (1-stage) showed relatively high levels of cellular membrane fluidity, caused by increased content in unsaturated fatty acids, compared to the conventional culture strategy (2-stage). Moreover, when this process was recycled by repeated-batch fermentation, the EPA productivity of 1-stage was 4.338 mg L-1 d-1, conspicuously increased by over 1300% compared to 2-stage. This strategy enhances the valuable omega-3 production, which can be commercially used for mass cultivation of omega-3-enriched biomass in the microalgae industry.