Background: Currently, it is not known how much walking should be advocated for good health in an adolescent population. Step count recommendations for minimum time in moderate-intensity activity have been translated predominantly from treadmill walking. Purpose: To compare the energy cost of walking on a treadmill with overground walking in adolescent girls. Methods: A total of 26 adolescent girls undertook resting metabolic measurements for individual determination of 1 metabolic equivalent using indirect calorimetry. Energy expenditure was subsequently assessed during treadmill and overground walking at slow, moderate, and fast walking speeds for 4 to 6 minutes. Treadmill step rates were matched overground using a metronome. Results: The energy cost of treadmill walking was found to be significantly greater than and not equivalent to overground walking at 133 steps per minute; (equivalent to the fast walking pace): $\dot{\mathrm{V}}{\mathrm{O}}_2$ 3.90 (2.78–5.01), P < .001, mean absolute percentage error (MAPE) = 18.18%, and metabolic equivalent 0.77 (0.54–1.00), P < .001, MAPE = 18.16%. The oxygen cost per step ($\dot{\mathrm{V}}{\mathrm{O}}_2$ mL·step⁻¹) was significantly greater and not equivalent on the treadmill at 120 and 133 steps per minute: 0.43 (0.12–0.56), P < .05, MAPE = 10.12% versus 1.40 (1.01–1.76), P < .001, MAPE = 17.64%, respectively. Conclusion: The results suggest that there is a difference in energy cost per step of walking on a treadmill and overground at the same step rate. This should be considered when utilizing the treadmill in energy expenditure studies. Studies which aim to provide step recommendations should focus on overground walking where most walking activity is adopted.