Disease-free sugarcane plantlets derived from meristem cutting were photoautotrophically grown on the MS medium and subsequently exposed to 0, 100, 200, 300 or 400 mM mannitol for 7 days. Osmotic pressure in the culture medium was increased with increase in mannitol concentration, causing low water use efficiency (WUE) (r2 = 0.88) and chlorophyll degradation (r2 = 0.92). Chlorophyll a (Chla), chlorophyll b (Chlb) and total carotenoids (Cx+c), concentrations in the osmotic stressed leaves decreased, especially in 400 mM mannitol treatment, degrading 44, 81 and 72%, respectively when compared to control. In contrast, proline content in osmotic stressed plantlets was accumulated and peaked at 2,236.75 μmol g-1 FW in 300 mM mannitol treatment. The WUE and chlorophyll degradation were correlated with maximum quantum yield of PSII (Fv/Fm) (r2 = 0.75) and photon yield of PSII (ΦPSII) (r2 = 0.83), respectively. The Fv/Fm and ΦPSII in drought acclimatized plantlets decreased, when non-photochemical quenching (NPQ) reached. The reduction of ΦPSII was positively related to net-photosynthetic rate (NPR) (r2 = 0.85) as well as the proline content and NPQ (r2 = 0.81). The NPR, stomatal conductance (Gs) and transpiration rate (E) in osmotic stressed plantlets were significantly dropped, leading to growth reduction (r2 = 0.95). The basic knowledge of osmotic stressed responses may further be applied as effective indices for drought tolerance in sugarcane breeding program.