Abstract I. Background: Estrogen receptor alpha (ERα) is a frequently mutated gene in breast cancer (BC). While many studies have investigated molecular dysregulation by hotspot mutations at Y537 and D538, which exhibit an estrogen-independent constitutively active phenotype, the functional abnormalities of other mutations remain obscure. The K303R mutation in primary invasive BC has been implicated with endocrine resistance, tumor size, and lymph node positivity. However, the impact of the K303R mutation on the cell epigenome is yet unknown. II. Methods and Results: We introduced the K303R ERa mutant in ERa-negative MDA-MB-453 cells to monitor ERa-dependent transactivation and to perform epigenomic analyses. The combination of ATAC-seq and ChIP-seq analyses indicated that both wild-type (WT) and the K303R mutant associated with Fox motif region at similar rates, even without an ERa-binding sequence, but only the K303R mutant induced chromatin opening at those regions. Biochemical analyses demonstrated that the WT and the K303R mutant forms of ERa can be tethered by Fox indirectly, but only the Fox/K303R/DNA complex can induce associations with the nuclear receptor cofactor NCOA2. III. Conclusions: These findings suggest that the K303R mutant induces chromatin opening at the Fox binding region through associations with NCOA2 and then probably disrupts regulation of Fox-target genes, resulting in K303R-related BC events.