Omecamtiv mecarbil (OM) is a myosin activator agent developed for the treatment of heart failure. OM was reported to increase left ventricular ejection fraction and systolic ejection time, but little is known about the effect of heart rate on the action of OM. The present study, therefore, was designed to investigate the effects of OM on unloaded cell shortening and intracellular Ca²⁺ ([Ca²⁺]_i) transients as a function of the pacing frequency. Isolated cardiomyocytes were stimulated at various frequencies under steady-state conditions. Cell length was monitored by an optical edge detector and changes in [Ca²⁺]_i were followed using the Ca²⁺-sensitive dye Fura-2. At the pacing frequency of 1 Hz, OM (1–10 μM) significantly decreased both diastolic and systolic cell length, however, fractional shortening was augmented only by 1 μM OM. Time to peak tension and time of 90% relaxation were progressively increased by OM. At the frequency of 2 Hz, diastolic cell length was reduced by 10 μM OM to a larger extent than systolic cell length, resulting in a significantly decreased fractional shortening under these conditions. OM had no effect on the parameters of the [Ca²⁺]_i transient at any pacing frequency. The results suggest that supratherapeutic concentrations of OM may decrease rather than increase the force of cardiac contraction especially in tachycardic patients.