Objective: Previous studies suggest that atrial natriuretic peptide (ANP) may act as an autocrine/paracrine factor to modulate cardiac hypertrophy in response to various stimuli. The effect of ANP deficiency on the response to volume overload has not previously been studied. We hypothesised that ANP deficient mice would develop excess cardiac hypertrophy in response to volume overload stress. Methods: Male homozygous ANP deficient (Nppa^−/−) and wildtype (Nppa^+/+) male mice maintained on either a normal salt (0.55% NaCl) or low salt (0.05% NaCl) diet from weaning were studied after 2 weeks of volume overload from an aorto-caval fistula (ACF). Unoperated littermates served as controls. Left ventricular (LV) structure and function was evaluated by echocardiography. Heart, LV, and lung weights were determined at sacrifice. Myocyte diameter was measured by morphometric analysis of fixed sections of the left ventricle. Results: BP, heart weight, and LV weight were increased in Nppa^−/− vs. Nppa^+/+ unoperated mice. Nppa^−/− mice developed exaggerated heart and LV weight compared to Nppa^+/+ mice following ACF. Increased myocyte diameter paralleled increased echo LV wall thickness following ACF in Nppa^+/+ but not Nppa^−/− mice fed with 0.55% NaCl, indicating that an alternate mechanism contributed to increased wall thickness in Nppa^−/− mice. Mid-wall shortening was mildly depressed in the Nppa^−/− vs. Nppa^+/+ genotype following ACF with fed 0.55% NaCl. A 0.05% NaCl diet from weaning normalized BP, but did not prevent exaggerated cardiac enlargement and LV hypertrophy following ACF in Nppa^−/− mice. Conclusions: ANP-deficient mice exhibited an exaggerated increase in heart and LV weight in response to volume overload, which was not prevented by normalization of blood pressure. The findings suggest that ANP is an important physiologic modulator of the cardiac hypertrophy induced by volume overload.