Annexin 6 is one of a widely expressed family of calcium‐binding proteins found in most mammalian tissues, including the heart. Several studies have implicated annexin 6 in the regulation of intracellular Ca²⁺ signaling, and it has been shown in vitro to act as a modulator of the sarcoplasmic reticulum Ca²⁺‐release channel, cardiac L‐type calcium channel, and Na⁺/Ca²⁺ exchanger. To investigate the role of annexin 6 in intact cardiomyocytes, we used mice containing a targeted disruption of the annexin 6 gene. Compared with controls, the myocytes of annexin 6 null‐mutant mice demonstrated a significant increase in the rates of shortening and relengthening. Intracellular Ca²⁺ transients in fura‐2‐loaded cardiomyocytes induced by caffeine showed a normal baseline and amplitude, whereas the rate of decay was doubled in annexin 6^/‐myocytes compared with control mice. These results show that annexin 6 knockout in the mouse leads to an increase in myocyte contractility and faster diastolic Ca²+ removal from the cytoplasm. In light of published findings showing annexin 6 to be down‐regulated in end‐stage heart failure, these results are consistent with a role for annexin 6 as a negative inotropic factor in the regulation of cardiomyocyte mechanics.