To study the influence of different relative distances of guide vanes on the pressure pulsation characteristics of a S-shaped extension tubular pumping system, three-dimensional steady calculation and unsteady calculation were applied under 4 different relative positions of guide vanes by using ANSYS CFX software and the reliability of the numerical simulation was verified by a model test. The results show that the pump head and efficiency increase first and then decrease with the increasing of the guide vanes’ relative distance, and the pump head can be affected much more obviously. The relative distance of the guide vanes has no influence on the dominant frequency of the monitoring points, but it has greater impact on the amplitude. With an increase of the relative distance at the guide vanes’ inlet position, the amplitudes of dominant frequency and cross section area of high pressure pulsation strength can decrease. The pressure pulsation strength variation near the flange side is more obvious than that near the hub side. The amplitude of the dominant frequency decreases first and then increases at the guide vanes’ outlet and the cross-section area of high pressure fluctuation strength decreases first and then increases. The performance of the pump is optimum when the relative position of the guide vanes is set to be 16 mm.