Saline-alkaline (SA) stress is a complex effect of three main factors: ion toxicity from high salinity, alkalinity, and high osmotic pressure. In this study, we dissected response of rice (Oryza sativa L.) to above-mentioned SA stress factors simulated by using NaCl, Na 2 CO 3 , and PEG6000, respectively. Seed germination was most strongly affected by osmotic stress, followed by alkaline and salt stresses. Meanwhile, the seedling growth was most reduced under alkaline stress. The alkaline treatment significantly reduced total biomass, total root length (TRL), root surface area (RSA), root numbers (RN), and root volume (RV), while it increased root diameter (RD) and Na + /K + ratio. Further, it caused severe damages to the root system as shown by a remarkable increase in degree of cell injury and expression of the cell death-related gene OsNAC4. On the other hand, salt stress reduced the total biomass, RSA and RV, and increased the Na + /K + ratio, while to less extent than those under alkaline stress. These results suggest that the deleterious effect of alkaline stress on rice seedlings is due mainly to high alkalinity that induces cell injury in the root system. In contrast, the response to osmotic stress exhibited the typical adaptive responses to dehydration: reduced relative water content (RWC), RD, RV, and shoot biomass; whereas increased TRL, RSA, RN, and root biomass. In addition, we show that proline was accumulated most abundantly in response to alkaline treatment, and which was corroborated by the transcriptional induction of OsP5CS1 and OsP5CS2, the genes encoding proline carboxylate synthase.