The large water footprint is one of the major bottlenecks for the sustainable microalgae-based biorefinery. In order to reduce the amount of water that is needed for mass cultivation of microalgae, the reusability of culture medium for further algal growth was tested. Chlorella vulgaris was cultivated in recycled medium that was obtained from harvesting microalgal cells by using either centrifugation or flocculation with FeCl3 or alum. The present study shows that centrifugation and flocculation with FeCl3 are equally effective (>90%) for harvesting C. vulgaris without any deleterious effects on algal growth when recycled media was used. However, even low concentration (<5 ppm) of residual alum was shown to inhibit microalgal growth. More interestingly, the recycled media obtained after centrifugation or flocculation with FeCl3 had a positive effect on biomass and lipid productivity of C. vulgaris. Extracellular substances such as carbohydrate, proteins, or ferric ions in the recycled media appear to cause these positive effects. Furthermore, change of pH to 2-3 and washing with water were found to effectively remove the residual ferric ions that are present in either harvested biomass or biodiesel, respectively. These results suggest that the use of recycled medium for microalgal cultivation is possible, and the choice of harvest methods must be carefully made when the recycling of culture medium is considered for microalgal cultivation.