Alternative splicing of the smooth muscle myosin phosphatase targeting subunit (Mypt1) exon 23 (E23) is tissue-specific and developmentally regulated and, thus, an attractive model for the study of smooth muscle phenotypic specification. We have proposed that Tra2β functions as a tissue-specific activator of Mypt1 E23 splicing on the basis of concordant expression patterns and Tra2β activation of Mypt1 E23 mini-gene splicing in vitro. In this study we examined the relationship between Tra2β and Mypt1 E23 splicing in vivo in the mouse. Tra2β was 2- to 5-fold more abundant in phasic smooth muscle tissues, such as the portal vein, small intestine, and small mesenteric artery, in which Mypt1 E23 is predominately included as compared with the tonic smooth muscle tissues, such as the aorta and inferior vena cava, in which Mypt1 E23 is predominately skipped. Tra2β was up-regulated in the small intestine postnatally, concordant with a switch to Mypt1 E23 splicing. Targeting of Tra2β in smooth muscle cells using SM22α-Cre caused a substantial reduction in Mypt1 E23 inclusion specifically in the intestinal smooth muscle of heterozygotes, indicating sensitivity to Tra2β gene dosage. The switch to the Mypt1 E23 skipped isoform coding for the C-terminal leucine zipper motif caused increased sensitivity of the muscle to the relaxant effects of 8-Br-cyclic guanosine monophosphate (cGMP). We conclude that Tra2β is necessary for the tissue-specific splicing of Mypt1 E23 in the phasic intestinal smooth muscle. Tra2β, by regulating the splicing of Mypt1 E23, sets the sensitivity of smooth muscle to cGMP-mediated relaxation.