Studies using a triaxial accelerometer and heart rate (HR) simultaneously for estimating energy expenditure (EE) during uphill exercise are rare. Exploring the optimal location for placing the accelerometer for predicting EE during uphill exercise is essential. Sixteen healthy male participants (M ± SEM; age 25.00 ± 0.61 years; body weight 74.13 ± 2.51 kg; body height 1.74 ± 0.01 m; body mass index 24.30 ± 0.63 kg/m2) exercised on a treadmill under 12 conditions (4 speeds and 3 gradients) on 3 days. Triaxial accelerometers, an HR recorder, and a metabolic measurement system were simultaneously used. Accelerometer outputs from various anatomical locations (upper arm, chest, lower back, waist, thigh, and instep) showed significant positive correlations with EE (0.819, 0.846, 0.816, 0.820, 0.672, and 0.669, respectively; p <.05). The linear regression equation for changes in HR showed the highest coefficient of determination (r2) of.837 with 87.9% reliability. When the HR signal was included, the r2 value (>.842) and reliability (87.9%) between the accelerometer outputs and EE improved. Accelerometer outputs from the waist position alone provide highly accurate EE values. Using both accelerometer outputs and HR for EE estimation during uphill exercise is feasible and improves the accuracy of EE prediction.