At the end of every heartbeat, cardiac myocytes must relax to allow filling of the heart. Impaired relaxation is a significant factor in heart failure, but all pathways regulating the cardiac relaxation apparatus are not known. Haploinsufficiency of the T-box transcription factor Tbx5 in mouse and man causes congenital heart defects (CHDs) as part of Holt–Oram syndrome (HOS). Here, we show that haploinsufficiency of Tbx5 in mouse results in cell-autonomous defects in ventricular relaxation. Tbx5 dosage modulates expression of the sarco(endo)plasmic reticulum Ca 2+ -ATPase isoform 2a encoded by Atp2a2 and Tbx5 haploinsufficiency in ventricular myocytes results in impaired Ca 2+ uptake dynamics and Ca 2+ transient prolongation. We also demonstrate that Tbx5 can activate the Atp2a2 promoter. Furthermore, we find that patients with HOS have significant diastolic filling abnormalities. These results reveal a direct genetic pathway that regulates cardiac diastolic function, implying that patients with structural CHDs may have clinically important underlying anomalies in heart function that merit treatment.