In addition to heat shock (HS), a proline analog, azetidine-2-carboxylic acid (Aze), also induces heat shock protein (HSP) synthesis. Here, we describe our further characterization of the induced effects by Aze treatment in soybean seedlings. Northern blot analyses revealed transcription of the soybean HS genes hsp70 and hsc70 as well as hspl7.5 activated after 6 h of Aze incubation, as reported previously. However, Aze-induced activation of HSP genes was not attenuated after transferring seedlings pretreated with Aze to shaking buffer, whereas the activation was decreased when seedlings were treated with Aze and then proline. Besides triggering an HS-like response, Aze treatment triggered a continuous increase of cellular leakage in soybean seedlings at room temperature. Nonlethal HS pretreatment protected seedlings from cellular leakage induced by Aze and allowed them to survive subsequent lethal HS treatment. As well, 10 mM proline prevented electrolyte leakage after 6 h of 10 mM Aze treatment and increased the survival of seedlings under lethal treatment. These results indicate that Aze treatment causes a permeability change in the plasma membrane by leading to cellular leakage before accumulation of HSPs. In vitro chaperone activity of 10 mM Aze-induced sHSP-CIs (class I small HSPs) with the model substrate citrate synthase was less efficient than that from 2 h of 40 °C treatment.