Differences in physiology and gene expression between ATHK1 knock-out mutant caused by T-DNA insertion and wild type (WT) of WS accession of Arabidopsis thaliana were analysed. Water loss ratio of detached leaf of ATHK1-mutant was obviously higher than that of WT. After being treated with 30% PEG-6000, ion leakage ratio of cell membrane in wild type leaves was 50% higher than that before PEG treatment, while in mutant leaves it increased 80%. The wilted phenotype of ATHK1-mutant after PEG treatment for 48 h was higher than that of WT. All these results showed that ATHK1-mutant was more sensitive to osmotic stress compared to WT and ATHK1 involved in osmotic stress adaptation. Differential-Display Reverse Transcription-PCR (DDRT-PCR) analysis was carried out to investigate the difference of gene expression between ATHK1-mutant and WT. Nine differential cDNA fragments involved in stress adaptation were identified, including the MAPKKK18 and serine/threonine protein kinase genes. These fragments were up-regulated by PEG treatment in WT, but not in ATHK1-mutant. These results indicate that ATHK1 plays an important role up-stream from MAPK in the osmotic stress signal transduction pathway. ATHK1 may be working as a plant osmosensor.