Alternative splicing is responsible for generating new isoforms from a single gene. Multiple splice variants for the estrogen receptor α (ERα) with one or more skipped exons have been identified, some of which are translated to proteins with modified functions from the wild-type 66 kDa. We aimed to silence splicing factors SF3B1 and SRSF-1 using our technology of Polypurine Reverse Hoogsteen Hairpins (PPRHs) in breast cancer cells and to analyse the possible changes in ERα splicing pattern. Different PPRHs against the SF3B1 and SRSF-1 genes were designed, and their cytotoxic effect was evaluated in breast cancer cell lines in combination with tamoxifen, observing a higher cytotoxicity than with tamoxifen alone. Inhibition of SF3B1 or SRSF-1 affected the inclusion/skipping ratios of several ERα exons in the MCF-7 cell line and in ex vivo cell cultures from fresh breast tumour samples derived from PDX. These variations were also observed at the protein level through Western blot analyses. Furthermore, RNAseq data from cells transfected with both PPRHs suggest an important role of SF3B1 and SFSR1 in alternative splicing since 220 and 277 genes were significantly differentially spliced, respectively, especially within the exon skipping category. Interestingly, SRSF1 inhibition affected the alternative splicing of several transcription factors, cyclin D3 and AKT, out of the genes frequently mutated in breast cancer. Our results indicate a role for both SF3B1 and SRSF-1 in ERα splicing. Therefore, PPRHs directed against these splicing factors could be regarded as potential therapeutic agents in a combination therapy with tamoxifen.