The cAMP response element modulator (CREM) plays pivotal roles in the hypothalamic‐pituitary‐gonadal axis. CREM mRNA is robustly expressed in human myocardium, and identified isoforms may suppress cAMP response element‐mediated transcription. However, little is known about the physiological importance of CREM in intact hearts remains unknown. We studied CREM‐null mice and age‐matched control littermates by in vivo pressure‐volume loops to analyze basal and reserve cardiac function. Basal systolic and diastolic function, echocardiographic morphology, and myocardial histology were normal in CREM‐null animals. However functional reserve with increasing heart rate was markedly depressed, with less contractile augmentation (+22±9% CREM^−/− vs.+62±11% controls, P<0.05) and relaxation shortening (5±5% CREM^−/− vs. −18±3% controls; P<0.05) at faster rates. In contrast, isoproterenol dose‐responses were similar, suggesting normal β‐adrenergic receptor‐coupled signaling. Gene expression of calcium handling proteins (SERCA, phospholamban) and stress‐response genes (e.g., α‐skeletal actin, β‐myosin heavy chain, natriuretic peptides) were similar between groups. However, total and serine‐phosphorylated phospholamban protein declined −38 and −64% respectively, and protein phosphatase‐1 (PP1) activity increased 44% without increased protein levels (all P<0.01) in CREM^−/− vs. controls. These results demonstrate novel involvement of CREM in regulation of PP1 activity and of PLB, likely resulting in a potent frequency‐dependent influence on cardiac function.— Isoda, T., Paolocci, N., Haghighi, K., Wang, C., Wang, Y., Georgakopoulos, D., Servillo, G., Della Fazia, M. A., Kranias, E. G., DePaoli‐Roach, A. A., Sassone‐Corsi, P., Kass, D. A. Novel regulation of cardiac force‐frequency relation by CREM (cAMP response element modulator). FASEB J. 17, 144–151 (2003)