(1) Background: Forward step-up (FSU) simulates the stance phase in stair ascension. With the benefits of physical properties of water, aquatic FSU exercise may be more suitable for patients with lower limb weakness or pain. The purpose of this study is to investigate the effect of progressive steps per min on the surface electromyography (sEMG) of gluteus maximus (GM), biceps femoris (BF), rectus femoris (RF), and gastrocnemius (GA), when performing FSU exercise with different steps per min in water and on land. (2) Methods: Participants (N = 20) were instructed to perform FSU exercises at different steps per min (35, 60, and 95 bpm) in water and on land. The sEMG of the tested muscles were collected. The percentage maximum voluntary isometric contraction (%MVIC) of GM, RF, GA and BF at different environments and steps per min was compared. (3) Result: There was a statistically significant difference of %MVIC of RF at all steps per min comparisons regardless of the movement phases and environments (p < 0.01, except for descending phases of 35 bpm vs. 60 bpm). All tested muscles showed a statistically significant lower muscle activation in water (p < 0.05) (4) Conclusion: This study found that the %MVIC of the tested muscle in both investigated environments increase as steps per minute increases. It is also found that the movement pattern of FSU exercise activates RF the most among all the tested muscles. Muscle activation of all tested muscles is also found to be smaller in water due to buoyancy property of water. Aquatic FSU exercise might be applicable to patients with lower limb weakness or knee osteoarthritis to improve their lower limb strength.