Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-α (TNF-α), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca²⁺ (Ca) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of β-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by ≈20%). The amplitude of Ca transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD_50%) of the Catransient, an index of the rate of sarcoplasmic reticulum Ca²⁺ uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca transients and contractions were reduced, TD_50% of the Ca transient was prolonged, and the inotropic effect of Iso on Ca transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca²⁺-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca²⁺ handling in the former. These results indicate that cardiac-specific overexpression of TNF-α induces myocyte hypertrophy and gender-dependent alterations in Ca handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.