Highlights•ZRSR1mu produces severe defects in erythrocytes, muscle stretch, and spermatogenesis•Spermatogenesis defects of ZRSR1mu are due to splicing alteration in U12-type intron•ZRSR1mu causes retention of most of the U12-type introns examined•ZRSR1 is implicated also in removal of U2 introns positioned adjacent to a U12 intronSummaryThe U2AF35-like ZRSR1 has been implicated in the recognition of 3′ splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.Graphical abstract