In order to explore the physiological acclimation of Gracilariopsis lemaneiformis to hypo-osmotic stress, in this study, the morphogenesis, photosynthetic physiological responses and ultrastructure changes of algal fragments from the main branches after being cultivated at combinations of four different low salinities (S 19.500, S 13.000, S 6.500 and S 0.000) and for five different periods (1, 3, 6, 12 and 20 h) and then cultured at normal salinity for 28 days were analysed. The results showed that less than 12 h hypo-osmotic stress treatment could promote the regeneration of buds in algal fragments of G. lemaneiformis. After 3 hours of hypo-osmotic stress with freshwater, increased fresh weight and number of regenerated buds obviously emerged in these fragments during normal salinity of culture. The relative growth rate (RGR) of algal fragments treated with freshwater for 3 hours was 0.91%/d, which was 61.27% higher than that of the control group. Furthermore, 1 h or 3 h hypo-osmotic stress with freshwater had no significant negative effects on the photosynthetic physiological responses of algal fragments, manifesting their photosynthetic activity was stimulated after restoration to seawater at normal salinity. It suggested that the cell metabolic activity of algal fragments might be enhanced after hypo-osmotic stress with freshwater. Ultrastructural observations showed that abundant floridean starch grains and plastoglobuli, sporadic lipid droplets emerged in the epidermal cell after 3 hours of hypo-osmotic stress with freshwater which would provide metabolic energy and membrane components for the algal fragments upon budding regeneration. On the contrary, long-term hypo-osmotic stress with freshwater caused irreversible structural damages to the organelles such as chloroplasts and thylakoids. These results indicated that the 3-hour treatment of hypo-osmotic stress with freshwater for algal fragments from main branches would stimulate their budding regeneration. This study would provide technical reference for rapid asexual propagations of G. lemaneiformis seedlings.