Changes of Selenoprotein F (SELENOF) protein levels have been reported during selenium supplementation, stressful, and pathological conditions. However, the mechanisms of how these external factors regulate SELENOF gene expression are largely unknown. In this study, HEK293T cells were chosen as an in vitro model. The 5'-flanking regions of SELENOF were analyzed for promoter features. Dual-Glo Luciferase assays were used to detect promoter activities. Putative binding sites of Heat Shock Factor 1 (HSF1) were predicted in silico and the associations were further proved by chromatin immunoprecipitation (ChIP) assay. Selenate and tunicamycin (Tm) treatment were used to induce SELENOF up-regulation. The fold changes in SELENOF expression and other relative proteins were analyzed by Q-PCR and western blot. Our results showed that selenate and Tm treatment up-regulated SELENOF at mRNA and protein levels. SELENOF 5'-flanking regions from -818 to -248 were identified as core positive regulatory element regions. Four putative HSF1 binding sites were predicted in regions from -1430 to -248, and six out of seven primers detected positive results in ChIP assay. HSF1 over-expression and heat shock activation increased the promoter activities, and mRNA and protein levels of SELENOF. Over-expression and knockdown of HSF1 showed transcriptional regulation effects on SELENOF during selenate and Tm treatment. In conclusion, HSF1 was discovered as one of the transcription factors that were associated with SELENOF 5'-flanking regions and mediated the up-regulation of SELENOF during selenate and Tm treatment. Our work has provided experimental data for the molecular mechanism of SELENOF gene regulation, as well as uncovered the involvement of HSF1 in selenotranscriptomic for the first time.