Abstract The effect of phase shift between serial-connected two pump chambers on the pumping performance of valveless micropumps has been investigated. Inspired by the two-pump organs of mosquitoes, two different valveless micropumps consisting of serial-connected two-pump chambers and three diffuser elements were fabricated using the conventional MEMS fabrication process. The two-pump chambers of the type 1 pump (TP I) have the same size, whereas those of the type 2 pump (TP II) have a different area. The volume flow rate was measured by varying the operating frequency and the phase shift by using a specially designed control system. The pumping performance of the serial-connected two micropumps is heavily dependent on the phase shift. The optimum phase shifts of both micropumps are 180° out-of-phase at high operating frequencies. When the operating frequency was decreased below 500Hz, they became closer to in-phase (θ=0°) condition. The phase shift has a significant influence on the pumping performance which is increased 186% at the pumping frequency of 1.1kHz. The results of this study are helpful in understanding the optimum operation of a serial-connected double-chamber micropump.