The crucial zeroth-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 zeroth-order light caused by the pixelation effect of SLM can be significantly eliminated. The method is further validated in an inverted fluorescence microscope. The experimental results show that the zeroth-order light can be inhibited up to 91.3%, accompanied by an improvement of the modulation efficiency from 77.5% to 92.6%.