The aggregation of nanoscale zero-valent iron (nZVI) particles and their limited transport ability in environmental media hinder their application in environmental remediation. In this study, the Cr(VI) removal efficiency, transport performance, and toxicity of nZVI and bentonite-modified nZVI (B-nZVI) were investigated. Compared with nZVI, B-nZVI improved the removal efficiency of Cr(VI) by 10%, and also significantly increased the transport in quartz sand and soil. Increasing the flow rate can enhance the transport of nZVI and B-nZVI in the quartz sand columns. The transport of the two materials in different soils was negatively correlated with the clay composition. Besides, modification of nZVI by bentonite could reduce toxicity to luminous bacteria (Photobacterium phosphereum T3) and ryegrass (Lolium perenne L.). Compared with Fe-EDTA, the transfer factors of nZVI and B-nZVI were 65.0% and 66.4% lower, respectively. This indicated that although iron nanoparticles accumulated in the roots of ryegrass, they were difficult to be transported to the shoots. The results of this study indicate that B-nZVI has a strong application potential in in situ environmental remediation.