The function of cytosolic AtHsp90.3 was characterized by complementing the Saccharomyces cerevisiae endogenous Hsp90 genes and overexpressing it in Arabidopsis thaliana. Though AtHsp90.3 supported the yeast growth under heat stress, in Arabidopsis, compared to the wild type, the transgenic plants overexpressing cytosolic AtHsp90.3 were more sensitive to heat stress with a lower germination rate and higher mortality but and more tolerant to high Ca2+. Transcriptional expression of heat stress transcription factors, AtHsfA1d, AtHsfA7a and AtHsfB1, and two Hsps, AtHsp101 and AtHsp17, was delayed by constitutive overexpression of cytosolic AtHsp90.3 under heat stress. These results indicate that overexpressing AtHsp90.3 impaired plant tolerance to heat stress and proper homeostasis of Hsp90 was critical for cellular stress response and/or tolerance in plants.