The rapid spread of antibiotic resistance genes (ARGs) in the environment is a major global concern to the public health. Although antibiotic resistance is considered as a natural mechanism of microorganisms before synthetic antibiotic usages, there is little knowledge about biological factors, especially predation, as a potential driver of antibiotic resistance in natural habitats. Soil protists are a pivotal component of soil food webs and primary microbial predator of bacteria and fungi. Bacteria can produce secondary metabolites such as antibiotics to fight against the predation of protists, and thus it is hypothesized that predation pressure of protists may drive the development of ARGs in the natural environment. In laboratory microcosm incubation, we examined the effects of two widely used antibiotics, oxytetracycline and ciprofloxacin, on the protistan communities in an arable soil. We found that the antibiotics significantly reduced the relative abundance of the dominant supergroups of protistan consumers Rhizaria and Amoebozoa, and provided empirical evidence that antibiotics can influence the protistan communities in the soil environment. We further established a soil microcosm incubation with a gradient of three protist concentrations to unravel protists’ effects on the profile of ARGs. Our results indicate that the abundance and number of ARGs were significantly higher in high protist treatments, compared to low and medium treatments. Notably, higher protist concentrations significantly increased the abundance and number of dominant ARGs encoding main mechanisms of antibiotic resistance, including efflux pump and antibiotic deactivation. Moreover, we identified positive protist-bacterial and protist-ARG associations, and the increasing abundance of numerous bacterial genera, which indicate the enhancement of bacterial growth and activities induced by the higher pressure of protists. Our novel findings demonstrated that soil protists are an important factor to the development of the antibiotic resistance of bacterial community.