The crucial zero-order light due to the pixelation effect of spatial light modulator (SLM) has been a serious issue in the field of light modulation, especially in applications with a high numerical aperture optical system. In this investigation, we report that by properly adjusting the high-level and low-level pixel voltages of an SLM, the zero-order light caused by the pixelation effect of an SLM can be significantly eliminated. The method is further validated under an inverted fluorescence microscope. The experimental results show that the zero-order light can be inhibited up to 91.3%, accompanied by an improvement of the modulation efficiency from 77.5% to 92.6%.