To design, synthetic promoters leading to stress-specific induction of a transgene, the study of cis-regulatory elements is of great importance. Cis-regulatory elements play a major role in regulating the gene expression spatially and temporally at the transcriptional level. The present work focuses on one of the important cis-regulatory element, W-box having TGAC as a core motif which serves as a binding site for the members of the WRKY transcription factor family. In the present study, we have analyzed the occurrence frequency of TGAC core motifs for varying spacer lengths (ranging from 0 to 30 base pairs) across the Arabidopsis thaliana genome in order to determine the biological and functional significance of these conserved sequences. Further, the available microarray data was used to determine the role of TGAC motif in abiotic stresses namely salinity, osmolarity and heat. It was observed that TGAC motifs with spacer sequences like TGACCCATTTTGAC and TGACCCATGAATTTTGAC had a significant deviation in frequency and were thought to be favored for transcriptional bindings. The microarray data analysis revealed the involvement of TGAC motif mainly with genes down-regulated under abiotic stress conditions. These results were further confirmed by the transient expression studies with promoter-reporter cassettes carrying TGAC and TGAC-ACGT variant motifs with spacer lengths of 5 and 10.