Abstract Body (Do not enter title and authors here): DHX38, a DEAH-box RNA helicase critical for pre-mRNA splicing, plays an essential role in maintaining transcriptomic fidelity, yet its function in the heart remains poorly characterized. Here, we define the molecular and physiological impact of a pathogenic missense mutation in DHX38 (p.Thr1221Met). Structural modeling and molecular dynamics simulations predict that the mutation induces localized conformational rigidity without gross destabilization of the helicase. Using a knock-in mouse model, multi-omics profiling revealed widespread transcriptomic and splicing dysregulation affecting genes involved in Rho GTPases, nonsense-mediated decay, and mitochondrial function. Notably, MYC signaling emerged as a disrupted regulatory axis, with suppression of canonical MYC targets involved in ribosome biogenesis, glycolysis, and cell cycle control. Proteomic analysis confirmed corresponding changes, including alterations in Rho GTPase regulators, rRNA processing proteins, and sarcomeric components, alongside upregulation of cardiac stress markers (e.g., Nppa, Postn, Mmp2, Fhl1/2). Functionally, homozygous Dhx38^{T1221M/T1221M} mice exhibited diastolic dysfunction despite preserved systolic function, as assessed by echocardiography. Collectively, these data demonstrate that DHX38 maintains cardiac homeostasis by coordinating splicing fidelity with transcriptional and translational control. Disruption of this axis by the p.Thr1221Met variant impairs MYC activity and RNA maturation, leading to diastolic dysfunction. These findings establish DHX38 as a central regulator of cardiac molecular integrity and highlight spliceosome dysfunction as a potential driver of cardiomyopathy.