In eukaryotes, alternative splicing plays an important role in proteome diversity and sound protein expression. Polypyrimidine tract binding protein (PTB) is a major alternative splicing factor that has been involved mostly with splicing repression. It has been proposed that the mechanism of action of PTB involves a looping out of exons flanked by pyrimidine tracts. Here, we present Fluorescence, NMR and single molecule data that directly support this mechanism. We show that the RNA binding domains 3 and 4 of PTB (RBD34) bind two distant pyrimidine‐tracts and can bring their 5' and 3' ends in close proximity, thus looping the RNA. Looping efficiency depends on the length of the intervening sequence with preference for a 15‐nucleotide spacer between the pyrimidine‐tracts. NMR chemical shifts perturbation show a specific directionality of the PTB inter‐action with RNA, RRM3 and RRM4 binding the 5' and the 3' pyrimidine‐tract, respectively. A PTB mutant reveals a synergistic effect between RBD3 and 4 for efficient looping in vitro and repress splicing in vivo.