Road dust is considered an important source of sediment contamination; however, there are few studies on the toxicity of road dust on benthic organisms. This study evaluates the toxicity of road dust on the benthic ostracod, Heterocypris incongruens, through a 6-day direct exposure experiment. All six road dust samples collected in heavy traffic areas caused high mortality of the ostracod, whereas the road dust collected in a residential area did not show toxicity to the ostracod. After maintaining the mixture of road dust and water for 24 h, the toxicity of the road dust decreased significantly for three of the six samples in the heavy traffic areas, suggesting these road dust samples become less toxic in the surface runoff process. In addition, we conducted the same toxicity test on manipulated road dust using different solid/liquid ratios and holding times to evaluate the change in toxicity caused by the runoff process. Wet road dust that had been separated from a dust-water mixture after a holding time of 1 h or 24 h did not show lethal toxicity, while the water-soluble fraction of the mixture caused high mortality of the ostracod at a solid/liquid ratio of 1:2 and 1:4. However, after a 7-day holding time of the dust-water mixture, the wet road dust and the water-soluble fraction showed lethal toxicity to the ostracod. These results suggest that toxicants of road dust seemed to exist mainly in water soluble fraction eluted off by rain water; however, particle-bound fraction again showed lethal toxicity after 7 days of incubation.