Development of bio-microactuators combining microdevices and cellular mechanical functions has been an active research field owing to their desirable properties including high mechanical integrity and biocompatibility. Although various types of devices were reported, the use of as-is natural muscle tissue should be more effective. An earthworm muscle-driven valve has been created. Long-time (more than 2 min) and repeatable displacement was observed by chemical (acetylcholine) stimulation. The generated force of the muscle (1 cm × 3 cm) was 1.57 mN on average for 2 min by the acetylcholine solution (100 mM) stimulation. We demonstrated an on-chip valve that stopped the constant pressure flow by the muscle contraction. For electrical control, short pulse stimulation was used for the continuous and repeatable muscle contraction. The response time was 3 s, and the pressure resistance was 3.0 kPa. Chemical stimulation was then used for continuous muscle contraction. The response time was 42 s, and the pressure resistance was 1.5 kPa. The ON (closed) state was kept for at least 2 min. An on-chip valve was demonstrated that stopped the constant pressure flow by the muscle contraction. This is the first demonstration of the muscle-based valve that is 100% chemically actuated and controlled.