Oocystis borgei, a microalgae species employed for regulating the quality of aquaculture water, demonstrates the capacity to adsorb noxious substances, curtail the growth of detrimental bacteria, and outcompete blooming cyanobacteria. It can be concentrated by natural sedimentation and stored at room temperature, making it costless and simple to transport and use. To study the mechanism of adaptation to room temperature preservation, O. borgei was concentrated (1.19 × 107-1.21 × 107 cell/mL) and stored for 50 days at low (5 °C, LT), normal (25 °C, NT), and high (35 °C, HT) temperatures, respectively. Polysaccharide content, lipid content, cell survival, and resuscitation were evaluated. RNA-Seq was also used to examine how concentrated O. borgei responded to temperature. During storage, there was an increase in polysaccharide content and a decrease in lipid content, with both being significantly upregulated in the LT and HT groups. Survival and cell density were highest in the NT group. The RNA-Seq analysis revealed extensive differences in transcript levels. ATP synthesis was inhibited in the LT group due to the reduced expression of PsaD, PsaE, PsaF, PsaK, and PsaL. Under HT, the formation of reactive oxygen species (ROS) was facilitated by low levels of redox-related genes (nirA) and high levels of oxidative genes (gdhA, glna, and glts). The findings suggest that storing concentrated O. borgei at room temperature is optimal for microalgae preservation, enhancing theoretical research in this field. Our study provides further theoretical and practical support for the development of O. borgei as a live ecological preparation for aquaculture microalgae ecology management.