Glycine betaine (GB) can enhance heat tolerance and the accumulation of heat-shock protein (HSP) in plants, but the effects of GB on HSP accumulation during salt stress were not previously known. To investigate the mechanism of how GB influences the expression of HSP, wild-type tobacco (Nicotiana tabacum) seedlings pretreated with exogenous GB and BADH-transgenic tobacco plants that accumulated GB in vivo were studied during NaCl stress. A transient Ca2+ efflux was observed in the epidermal cells of the elongation zone of tobacco roots after NaCl treatment for 1–2 min. After 24 h of NaCl treatment, an influx of Ca2+ was observed; a low concentration of GB significantly increased NaCl-induced Ca2+ influx. GB increased the intracellular free calcium ion concentration and enhanced the expression of the calmodulin (CaM) and heat-shock transcription factor (HSF) genes resulting in potentiated levels of HSPs. Pharmacological experiments confirmed that Ca2+ and CaM increased HSFs and HSPs gene expression, which coincided with increased the levels of HSP70 accumulation. These results suggest a mechanism by which GB acted as a cofactor in the NaCl induction of a Ca2+-permeable current. A possible regulatory model of Ca2+-CaM in the signal transduction pathway for induction of transcription and translation of the active HSPs is described.