Provided are an A138T mutation-containing plant EPSPS mutant, and an encoding gene and an application thereof. The site at position 138, corresponding to escherichia coli EPSPS, of the amino acid sequence of the plant EPSPS mutant has the mutation A>；T. The mutation significantly increases the resistance of different EPSPS mutants of various plants to glyphosate, and maintains the biological enzyme catalysis activity. A plant or recombinant strain transformed with the plant EPSPS mutant can grow normally in the presence of glyphosate. The plant EPSPS mutant may be used for genetically modified crop cultivation, and may also be used for cultivating glyphosate-resistant non-genetically modified plants, such as rice, tobacco, soybean, corn, wheat, cotton and sorghum.
