Artificial green tea extracts may enhance exercise-induced fat oxidation. Natural Matcha green tea consumption involves the ingestion of the powdered green tea leaves. We examined the effects of three weeks daily intake of Matcha green tea powder on substrate oxidation during moderate-intensity exercise in females. Females with a regular menstrual cycle (n  = 12, age: 28 ± 10 yr, body mass: 69 ± 17 kg, height: 163 ± 6 cm) volunteered to complete an incremental walking test to determine the individual moderate exercise intensity (four metabolic equivalent) for the subsequent 30-min treadmill walk. The study had a randomized placebo-controlled cross-over design with participants tested between day 9 and 11 of the menstrual cycle (follicular phase). Participants consumed 3x1 gram capsules of Matcha premium grade, (OMGTea Ltd, UK) per day for three weeks, with the final dose (1 gram) two hours before the 30-min walk (walking speed: 5.8 ± 0.4 km·h−1). Matcha had no effect on physiological responses (e.g. heart rate, placebo: 127 ± 14; Matcha: 124 ± 14 beats·min−1, p  = 0.154), but resulted in lower respiratory exchange ratio (placebo: 0.872 ± 0.040; Matcha: 0.839 ± 0.035) (p  = 0.033), higher fat oxidation by 35 ± 47% (placebo: 0.21 ± 0.08; Matcha: 0.26 ± 0.06 g·min−1) (p  = 0.034), and lower carbohydrate oxidation (placebo: 0.75 ± 0.21; Matcha: 0.60 ± 0.18 g·min−1) (p = 0.048) during the 30-min moderate-intensity walk. Energy expenditure was similar for both conditions. There was no significant correlation between body fat % and the absolute or relative change in Matcha-induced fat oxidation during exercise. Continuous intake of Matcha green tea effects exercise-induced metabolic responses by enhancing fat oxidation during moderate-intensity exercise in adult females, seemingly independent of body composition.