The heat shock transcription factor 1 gene (HSF1) plays a key role in the heat stress response. We previously found a single nucleotide polymorphism (SNP) in the 3'-untranslated region (g.4693G>T) of HSF1 that was related to thermo tolerance in Chinese Holstein cattle through association analysis. However, it is not known whether other SNPs also affect thermo tolerance.In this study a novel SNP, g.1451G>T, was identified by DNA sequencing and genotyped using creating restriction site-polymerase chain reaction methodology. The g.1451G>T polymorphic site met Hardy-Weinberg equilibrium (P > 0.05). Association analysis demonstrated that this SNP had no effect on thermo tolerance traits in Holstein cattle. Findings of the study compared to the analysis of g.4693 G>T further indicated that g.4693 G>T may play an important role in thermo tolerance, although the mechanism is not clear. RNA hybrid and Targetscan prediction showed that the minimum free energy hybridization of bta-miR-484 with HSF1 3'-UTR was -31.9 kcal/mol and g.4693 G>T was in the seed sequence of bovine HSF1 that binds to bta-miR-484. Analysis by Luciferase assay indicated that HSF1 expression was directly targeted by bta-miR-484 in HEK 293T cells, and the Rluc/luc ratio of wildtype (GG) was lower than that of the mutant (TT) (P T affects binding of HSF1 to bta-miR-484.