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Abstract Pathogenic variants in the human Factor VIII (F8) gene cause Hemophilia A (HA). Here, we investigated the impact of 97 HA-causing single-nucleotide variants on the splicing of 11 exons from F8. For the majority of F8 exons, splicing was insensitive to the presence of HA-causing variants. However, splicing of several exons, including exon-16, was impacted by variants predicted to alter exonic splicing regulatory sequences. Using exon-16 as a model, we investigated the structure–function relationship of HA-causing variants on splicing. Intriguingly, RNA chemical probing analyses revealed a three-way junction structure at the 3′-end of intron-15 (TWJ-3–15) capable of sequestering the polypyrimidine tract. We discovered antisense oligonucleotides (ASOs) targeting TWJ-3–15 partially rescue splicing-deficient exon-16 variants by increasing accessibility of the polypyrimidine tract. The apical stem loop region of TWJ-3–15 also contains two hnRNPA1-dependent intronic splicing silencers (ISSs). ASOs blocking these ISSs also partially rescued splicing. When used in combination, ASOs targeting both the ISSs and the region sequestering the polypyrimidine tract, fully rescue pre-mRNA splicing of multiple HA-linked variants of exon-16. Together, our data reveal a putative RNA structure that sensitizes F8 exon-16 to aberrant splicing.