Context: Muscle activation during aquatic treadmill (ATM) running has not been examined, despite similar investigations for other modes of aquatic locomotion and increased interest in ATM running. Objectives: The objectives of this study were to compare normalized (percentage of maximal voluntary contraction; %MVC), absolute duration (aDUR), and total (tACT) lower-extremity muscle activity during land treadmill (TM) and ATM running at the same speeds. Design: Exploratory, quasi-experimental, crossover design. Setting: Athletic training facility. Participants: 12 healthy recreational runners (age = 25.8 ± 5 y, height = 178.4 ± 8.2 cm, mass = 71.5 ± 11.5 kg, running experience = 8.2 ± 5.3 y) volunteered for participation. Intervention: All participants performed TM and ATM running at 174.4, 201.2, and 228.0 m/min while surface electromyographic data were collected from the vastus medialis, rectus femoris, gastrocnemius, tibialis anterior, and biceps femoris. Main Outcome Measures: For each muscle, a 2 × 3 repeated-measures ANOVA was used to analyze the main effects and environment–speed interaction (P ≤ .05) of each dependent variable: %MVC, aDUR, and tACT. Results: Compared with TM, ATM elicited significantly reduced %MVC (−44.0%) but increased aDUR (+213.1%) and tACT (+41.9%) in the vastus medialis, increased %MVC (+48.7%) and aDUR (+128.1%) in the rectus femoris during swing phase, reduced %MVC (−26.9%) and tACT (−40.1%) in the gastrocnemius, increased aDUR (+33.1%) and tACT (+35.7%) in the tibialis anterior, and increased aDUR (+41.3%) and tACT (+29.2%) in the biceps femoris. At faster running speeds, there were significant increases in tibialis anterior %MVC (+8.6−15.2%) and tACT (+12.7−17.0%) and rectus femoris %MVC (12.1−26.6%; swing phase). Conclusion: No significant environment–speed interaction effects suggested that observed muscle-activity differences between ATM and TM were due to environmental variation, ie, buoyancy (presumed to decrease %MVC) and drag forces (presumed to increase aDUR and tACT) in the water.