Mitogen-activated protein kinase kinase kinase (MEKK1) mediates activation of c-Jun NH₂-terminal kinase (JNK). Although previous studies using cultured cardiac myocytes have suggested that the MEKK1-JNK pathway plays a key role in hypertrophy and apoptosis, its effects in cardiac hypertrophy and apoptosis are not fully understood in adult animals in vivo. We examined the role of the MEKK1-JNK pathway in pressure-overloaded hearts by using mice deficient in MEKK1. We found that transverse aortic banding significantly increased JNK activity in Mekk1^+/+ but not Mekk1^–/– mice, indicating that MEKK1 mediates JNK activation by pressure overload. Nevertheless, pressure overload caused significant levels of cardiac hypertrophy and expression of atrial natriuretic factor in Mekk1^–/– animals, which showed higher mortality and lung/body weight ratio than were seen in controls. Fourteen days after banding, Mekk1^–/– hearts were dilated, and their left ventricular ejection fraction was low. Pressure overload caused elevated levels of apoptosis and inflammatory lesions in these mice and produced a smaller increase in TGF-β and TNF-α expression than occurred in wild-type controls. Thus, MEKK1 appears to be required for pressure overload–induced JNK activation and cytokine upregulation but to be dispensable for pressure overload–induced cardiac hypertrophy. MEKK1 also prevents apoptosis and inflammation, thereby protecting against heart failure and sudden death following cardiac pressure overload.