Transposable elements (TEs) are a significant component of the human genome, occupying about half of the intronic space. While intronic TEs are transcribed along with their host genes, they are rarely included in the final mRNA due to rapid splicing and degradation. However, TEs can create new introns or chimeric transcripts, and their rare occurrence suggests a robust splicing code that suppresses TE exonization. This study identifies SAFB proteins as key regulators that protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity. SAFB proteins bind to conserved adenosine-rich sequences in L1 elements, preventing their exonization. This dual role is achieved through the binding of SAFB proteins to L1 RNA, which is enriched in sense L1 elements and extinct Tigger DNA transposons. Depletion of SAFB proteins leads to increased L1 retrotransposition and altered gene expression, particularly in genes containing SAFB-bound TEs. SAFB proteins also prevent the activation of cryptic splice sites within TEs, which can lead to gene disruption. Additionally, SAFB proteins regulate giant protein-coding cassette exons, which are often upregulated in SAFB-depleted cells. The ancestral function of SAFB proteins is conserved in mice and Drosophila, suggesting evolutionary conservation. The study highlights the evolutionary pressure to maintain adenosine bias in TE ORFs and the role of SAFB proteins in limiting TE activity under stress conditions, with a notable decrease in expression during spermatogenesis.Transposable elements (TEs) are a significant component of the human genome, occupying about half of the intronic space. While intronic TEs are transcribed along with their host genes, they are rarely included in the final mRNA due to rapid splicing and degradation. However, TEs can create new introns or chimeric transcripts, and their rare occurrence suggests a robust splicing code that suppresses TE exonization. This study identifies SAFB proteins as key regulators that protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity. SAFB proteins bind to conserved adenosine-rich sequences in L1 elements, preventing their exonization. This dual role is achieved through the binding of SAFB proteins to L1 RNA, which is enriched in sense L1 elements and extinct Tigger DNA transposons. Depletion of SAFB proteins leads to increased L1 retrotransposition and altered gene expression, particularly in genes containing SAFB-bound TEs. SAFB proteins also prevent the activation of cryptic splice sites within TEs, which can lead to gene disruption. Additionally, SAFB proteins regulate giant protein-coding cassette exons, which are often upregulated in SAFB-depleted cells. The ancestral function of SAFB proteins is conserved in mice and Drosophila, suggesting evolutionary conservation. The study highlights the evolutionary pressure to maintain adenosine bias in TE ORFs and the role of SAFB proteins in limiting TE activity under stress conditions, with a notable decrease in expression during spermatogenesis.