Pneumocandin B-0, the precursor of the antifungal drug caspofungin, is a secondary metabolite of the fungus Glarea lozoyensis. In this study, we investigated the effects of mannitol as the sole carbon source on pneumocandin B-0 production by G. lozoyensis. The osmotic pressure is more important in enhancing pneumocandin B-0 production than is the substrate concentration. Based on the kinetic analysis, an osmotic stress control fed-batch strategy was developed. This strategy led to a maximum pneumocandin B-0 concentration of 2711mg/L with a productivity of 9.05mg/L/h, representing 34.67 and 6.47% improvements, respectively, over the best result achieved by the one-stage fermentation. Furthermore, G. lozoyensis accumulated glutamate and proline as compatible solutes to resist osmotic stress, and these amino acids also provided the precursors for the enhanced pneumocandin B-0 production. Osmotic stress also activated ROS (reactive oxygen species)-dependent signal transduction by upregulating the levels of related genes and increasing intracellular ROS levels by 20%. We also provided a possible mechanism for pneumocandin B-0 accumulation based on signal transduction. These findings will improve our understanding of the regulatory mechanisms of pneumocandin B-0 biosynthesis and may be applied to improve secondary metabolite production.