Folate is a B-vitamin required for DNA synthesis, methylation, and cellular division, whose deficiencies are associated with various disorders and diseases. Currently, most folic acid used for fortification is synthesized chemically, causing undesirable side effects. However, using folate-producing probiotics is a viable option, which fortify folate in situ and regulate intestinal microbiota. In this study, the folate production potential of newly isolated strains from raw milk was analyzed by microbiological assay. Latilactobacillus sakei LZ217 showed the highest folate production in Folic Acid Assay Broth, 239.70 ± 0.03 ng/μL. The folate produced by LZ217 was identified as 5-methyltetrahydrofolate. LZ217 was tolerant to environmental stresses (temperature, pH, NaCl, and ethanol), and was resistant to gastrointestinal juices. Additionally, the in vitro effects of LZ217 on human gut microbiota were investigated by fecal slurry cultures. 16S rDNA gene sequencing indicated that fermented samples containing LZ217 significantly increased the abundance of phylum Firmicutes and genus Lactobacillus, Faecalibacterium, Ruminococcus 2, Butyricicoccus compared to not containing. Short-chain fatty acids (SCFAs) analysis revealed that LZ217 also increased the production of butyric acid by fermentation. Together, L. sakei LZ217 could be considered as a probiotic candidate to fortify folate and regulate intestinal microecology.