The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2β1 to TRA2β5). TRA2B exon 2 encodes multiple premature termination codons. TRA2β1 lacks exon 2 and is translated into a functional transformer 2β (Tra2β) protein, whereas TRA2β4 contains 10 exons and works as a functional RNA. Overexpressed Tra2β and ectopic expression of TRA2β4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2β4 exon 2. Minigene assays revealed that hnRNPA1 facilitated inclusion of exon 2, whereas hnRNPU promoted its skipping. However, knockdown of hnRNPA1 or hnRNPU reduced both TRA2β1 and TRA2β4 levels, and overexpression of these hnRNPs increased levels of both isoforms, suggesting that hnRNPA1 and hnRNPU mainly regulate the transcription of TRA2B. In fact, hnRNPA1 and hnRNPU positively regulated the promoter activity of TRA2B. Circular dichroism analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated the presence of G-quadruplex (G4) formation in the promoter of TRA2B. Formation of G4 suppressed TRA2B transcription, whereas hnRNPA1, but not hnRNPU, interacted with the G4 to facilitate transcription. Our results suggest that hnRNPA1 may modulate TRA2B transcription through its regulation of G4 formation in its promoter in colon cancer cells.