Earthworms are invertebrates and contribute to soil fertility improvement and plant growth. Roundup is widely used herbicide in agriculture. Eisenia fetida has been suggested as a sensitive and standard species for ecotoxicological studies. Eisenia fetida were exposed to Roundup herbicide via soil. Acute toxicity evaluation of LC₅₀ and the effect of Roundup herbicide on the earthworm, Eisenia fetida was studied under laboratory conditions for 24, 48, and 72 hours and the result indicated the acute toxicity of Roundup herbicide concentrations was 0·050, 0·034 and 0·024 ppm to be specific respectively. The aim of the present investigation was to assess LC₅₀ values of Roundup herbicide was 0·04898, 0·03342 and 0·02350 and effect on survivality and mortality of earthworm, Eisenia fetida.