Nostoc flagelliforme is an endangered desiccation-tolerant cyanobacterium with dietary and herbal values. Enough availability of this biotic resource in food industry or for ecological management will come from the mass cultivation of its liquid suspension culture. Nostoc flagelliforme is sensitive to desiccation stress when cultivated as a cell suspension in liquid medium. In this study, we describe the desiccation tolerance acquisition of the liquid suspension culture on agar plates through slow drying treatment and investigate biochemical, proteomic, and ultrastructural changes of cells upon the drying induction. During the 16-day drying treatment, the cell suspension–derived solid cultures were maintained with 90 ~ 94% water content and the fresh weight increased nearly ninefold. Biochemical analysis showed that the biosynthesis of extracellular components, exopolysaccharide and scytonemin, was significantly increased in the solid cultures during the drying process. WspA protein, which can be secreted into the extracellular matrix, was also greatly induced. The contents of the compatible solutes trehalose and sucrose were not increased, but the content of trehalose maintained a high basal level. Proteomic analysis identified some highly upregulated proteins that are potentially crucial for desiccation tolerance, including WspA protein and Mn-containing catalase. Also, the biological processes regarding lipid or pigment metabolisms and the pathways regarding fructose and mannose metabolism and the biosynthesis of secondary metabolites were found among the most affected events during the drying process. Ultrastructural observation suggested that the structural modulation of the exopolysaccharide matrix occurred. In addition, the monosaccharide composition of exopolysaccharides was found to be specific for this liquid suspension culture or its derived solid culture. In general, this study provides insights into the mechanisms of desiccation tolerance acquisition in liquid suspension culture of N. flagelliforme and also proposes a new solution for developing desiccation-tolerant N. flagelliforme resource.