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This research aimed to determine the effects of Gynostemma pentaphyllum (<i>G. pentaphyllum</i>) on exercise performance, AMP-activated protein kinase (AMPK), and mitochondrial signaling in human muscle. This randomized double-blind placebo control crossover study provided placebo or 450 mg of <i>G. pentaphyllum</i> dried leaf extract equivalent to 2.25 g of dry leaf per day for four weeks to 16 healthy untrained young males, separated by four weeks wash-out. Following 4-week supplementation with <i>G. pentaphyllum,</i> participants had significantly lower leptin and blood glucose levels and improved time trial performance over 20 km, which corresponded with a higher muscle oxygen flux compared to placebo. Muscle AMPK Thr172 phosphorylation significantly increased after 60 min exercise following <i>G. pentaphyllum</i> supplementation. AMPK Thr172 phosphorylation levels relative to total AMPK increased earlier following exercise with <i>G. pentaphyllum</i> compared to placebo. Total ACC-α was lower following <i>G. pentaphyllum</i> supplementation compared to placebo. While further research is warranted, <i>G. pentaphyllum</i> supplementation improved exercise performance in healthy untrained males, which corresponded with improved mitochondrial respiration, altered AMPK and ACC, and decreased plasma leptin and glucose levels.