In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na+ contents increased, the retained K+ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na+-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.