The growth and productivity of cucumber are severely affected by salinity. To understand the complex salt response mechanism, the physiological and biochemical responses of cucumber seedlings to iso-osmotic NaCl and Ca(NO3)2 stresses were investigated. In this study, the biomass was significantly decreased under iso-osmotic NaCl and Ca(NO3)2 stresses, and the inhibitory effect of Ca(NO3)2 stress was less than that of NaCl stress. The soluble protein contents were increased under Ca(NO3)2 stress, whereas they were decreased after 6 days of NaCl stress. A sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that there were 14 differentially expressed protein bands in roots under iso-osmotic NaCl and Ca(NO3)2 stresses at 0, 3, 6, and 9 days, and seven protein bands were little expressed under NaCl stress at 6 and 9 days. Based on these results, 2-D gel electrophoresis was used to separate cucumber root proteins in response to iso-osmotic NaCl and Ca(NO3)2 stresses at 3 days. A total of 43 protein spots changed under salt stress. Of these proteins, 33 were successfully identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS) and categorized into classes, including those corresponding to antioxidants and defense-related proteins and energy and metabolism. The functions of the significantly differentially expressed root proteins were analyzed, which may facilitate a better understanding of different salt response mechanisms, and we suggest that cucumber seedlings showed a more powerful ability to resist Ca(NO3)2 stress.