A three-dimensional computational fluid dynamics simulation for a micro-scale pump has been performed to investigate the sensitivity of the flow rate to fre-quency change during pump and supply phases. Applying dynamic mesh under different diaphragm actuation frequencies (8, 50, 100, 200, and 500 Hz) and si-nusoidal oscillation amplitude of 6.0 µm the results showed that at frequency f  ≥ 100 Hz the flow separation has been formed in the diffuser direction for both supply and pump phases. At low frequency f ≤ 50 Hz there is no flow separation in the diffuser or the nozzle direction. The large recirculation zone is created at the diffuser bottom wall side. The flow separation was found to be frequency and direction dependent for the micro-diffuser half angle of 5o. The net flow rate increased linearly with the frequency while the net flow per cycle was less sen-sitive to the change of the frequency.