MiWRKY53 is expressed in response to various stresses and hormones. Although it is localized in the nucleus, it shows no transcriptional activation. Role of SA-mediated plant defence response is demonstrated. WRKY transcription factors are one the largest gene families in plants involved in almost every process in plants including development, physiological processes, and stress response. Salicylic acid (SA) is key regulator of biotic stress against various pathogens in plants acting via its multiple mechanisms to induce defence response. Herein, we have identified and functionally validated WRKY53 from mulberry (Morus indica var. K2). MiWRKY53 expressed differentially in response to different stress and hormonal treatments. MiWRKY53 belongs to group III of WKRY gene family, localized in nucleus, and lacks transcriptional activation activity in yeast. Hormone responsive behaviour of MiWRKY53 Arabidopsis overexpression (OE) transgenics preferentially was noted in root growth assay in response to Salicylic acid (SA). Arabidopsis overexpression plants also displayed alteration in leaf phenotype having wider leaves than the wild-type plants. PR-1 transcripts were higher in MiWRKY53 Arabidopsis OE plants and they displayed resistance towards biotrophic pathogen Pseudomonas syringae PstDC3000. MiWRKY53 Mulberry OE transgenics also depicted SA-responsive behaviour. Several hormones and stress-related cis-acting elements were also identified in the 1.2-Kb upstream regulatory region (URR) of MiWRKY53. Functional characterization of full-length promoter region revealed that it is induced by SA and further analysis of deletion constructs helped in the identification of minimal promoter responsible for its inducibility by SA. Altogether, the findings from this study point towards the SA preferential behaviour of MiWRKY53 and its function as regulator of plant defence response through SA-mediated mechanisms.