In this study, a novel composite of modified diatomite supported nanoscale zero-valent iron (mD-NZVI) was synthesized and characterized. The effects of four factors (mD-NZVI dose, temperature, contact time and initial pH) on the removal of Cr(VI) by mD-NZVI were studied by experimental work and analyzed by response surface methodology (RSM). A second-order polynomial equation fitted by Box-Behnken design was used as a statistical model and proved to be precise in describing the significance of four factors. The analysis results show that the effects of four factors on the removal efficiency of Cr(VI) were significant (F value is 19.83), initial pH was found to be the key factor. In addition, the effect of initial pH was further studied and the maximum removal efficiency of 89.34% was obtained at pH of 3, the decrease in removal efficiency with the increase in pH is a synergistic effect of Cr(VI) species, surface charge of mD-NZVI and OH− amount at different pH.