Among abiotic stresses, heat stress has the most devastating impact on plant growth. In the present study, heat shock transcription factor HsfA1d was isolated from Arabidopsis thaliana and cloned into an in-house constructed gateway compatible cloning vector pUC57GW-CmRccdB through infusion cloning. Entry clone was confirmed through restriction digestion and sequencing analysis. For subcellular localization, HsfA1d was cloned in-frame with yellow florescent protein (YFP) in pGWB442 through LR clonase reaction resulted in YFP-HsfA1d chimeric gene construct. Homodimerization of HsfA1d was studied using Bimolecular Florescence complementation (BIFC) assay. For BIFC, HsfA1d was cloned in-frame with sequences that codes for N and C termini of yellow florescent protein, into 2 in-house constructed vectors pGSA002-nYFPn and pGSA002-nYFPc respectively. After confirmation through restriction enzyme digestion, the newly constructed vectors were transformed into Agrobacterium tumefaciens GV3101 for transient expression in tobacco (Nicotiana benthamiana) through syringe-infiltration. Subcellular localization of YFP-HsfA1d in DAPI-stained cells was evaluated using confocal microscopy. For plant transformation, HsfA1d was cloned into 2 plant expression vectors pGWB402Ω and pGWB442 through LR clonase reaction. After confirmation through RFLP analysis, the vectors were transformed into Agrobacterium GV3101 for tobacco leaf disc infection. Putative transgenic plants were selected and regenerated on appropriate selection media using different growth regulators. After PCR and confocal-based confirmation, transgenic plants were evaluated for their response to heat stress. HsfA1d, HSP70 and HSP90 exhibited 4.8, 2.8 and 2 folds increase respectively in their expression under heat stress compare to room temperature. HsfA1d was found to significantly enhance the expression of downstream gene HSP70 while no such effect on the expression of HSP90 was recorded. The plants transformed with HsfA1d were found to retain more water and accumulate more proline under heat stress. The transgenic plants exhibited efficient protective system, causing less 8 electrolyte leakage and less chlorophyll damage under heat stress. It is concluded that HsfA1d plays a vital role in thermotolerance enhancement and hence recommended for increasing plant thermotolerance.