Lactic acid bacteria (LAB) are safe microorganisms that have been used in the processing of fermented food for centuries. The aim of this study was to isolate from fresh tea leaves and examine the impact of an isolated strain on intestinal barrier integrity. First, the presence of strains was investigated in fresh tea leaves from Kagoshima, Japan. Strains were isolated by growing on De Man, Rogosa and Sharpe (MRS) agar medium containing sodium carbonate, followed by the identification of one strain by polymerase chain reaction (PCR) and sequence analysis, with the strain identified as and named LOC1. Second, the impact of strain LOC1 in its heat-inactivated form on intestinal barrier integrity was investigated. Strain LOC1, but not ATCC 14917 or ATCC 8014, significantly suppressed dextran sulfate sodium (DSS)-induced decreases in transepithelial electrical resistance values of Caco-2:HT29-MTX 100:0 and 90:10 co-cultures. Moreover, in Caco-2:HT29-MTX co-cultures (90:10 and 75:25), levels of occludin mRNA were significantly increased by strain LOC1 compared with untreated co-cultures, and strain LOC1 had higher mRNA levels of MUC2 and MUC4 mucins than ATCC 14917 and YT9. These results indicate that LOC1 may be used as a safe probiotic with beneficial effects on the intestinal barrier, suggesting that fresh tea leaves could be utilized as a safe source for isolating probiotics.