Abstract: In this study, ¹²C⁶⁺ heavy ion beam released from heavy ion accelerator of Chinese Academy of Sciences was used as the radiation mutagenesis source. With acid spot value and bacteriostatic ring value as indexes, functional mutagenesis of Lactobacillus rhamnosus JF12-1 was carried out to determine the optimal radiation dose of mutagenesis through mortality rate and positive and negative mutation rate. The mutated strains under the optimal irradiation dose were screened by acid patch method and screened by inhibition zone method. Then the genetic stability of the genetically stable strains was determined by detecting the change of lactic acid content after continuous passage. 16S rDNA sequencing was performed to locate the mutation sites of the genetically stable strains. The results showed that the mortality rate, positive mutation rate and negative mutation rate were 79.86%, 30.33% and 5.38%, respectively, when the irradiation dose was 300 Gy, which was the best mutagenesis dose. The results showed that the HC value of 20 mutant strains increased by more than 25% compared with the original wild strain. The bacteriostatic activity of 8 strains increased by 15% compared with the original wild strains. Genetic stability analysis showed that the 8 mutant Lactobacillus strains were stable in producing lactic acid. 16S rDNA sequencing revealed that the mutated sites of JF12-1 were not in 16S rRNA gene, and the mutated sites promoting acid production and bacteriostatic performance of JF12-1 were probably in other gene segments. A functional Lactobacillus rhamnosus stable strain with high lactic acid yield and good bacteriostatic activity in vitro was successfully cultured by ¹²C⁶⁺ heavy ion beam mutation, which provided a good theoretical basis and application basis for further development of this strain.