Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects such as the regulation of AS and its functional implications largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis Polypyrimidine tract-binding protein homologues (PTBs) was revealed. In total 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5’ splice sites. In contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR 6, coinciding with altered rates of abscisic acid-dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in an PTB1/2 level-dependent manner.