Probiotics have a long history as fermented food or food supplements. The health benefits and safety profiles of probiotics are strain-specific and should be evaluated individually. The aim of this study was to assess the safety of the Lactobacillus plantarum GUANKE (GUANKE) strain by conducting pharmacological studies, oral toxicity assessments, and investigating the colonization and translocation of GUANKE in experimental animal models. Three pharmacological studies were conducted to examine the effects of oral administration of GUANKE on gastric emptying, bile secretion, and gastric juice secretion. In an acute toxicity study, rats were orally administrated with different doses of GUANKE and monitored for 14 days. In the subacute toxicity study, both rats and beagles were administrated with varying doses of GUANKE for 28 consecutive days to evaluate hematologic, biochemical, and histological effects. The results showed that GUANKE administration did not result in any adverse effect on hematological parameters, biochemical parameters, urinary parameters, and organ indices. Importantly, no translocation of GUANKE to extra-intestinal organs or blood was observed following administration of the CFDA-SE labeled strain. In summary, this study demonstrated the safety of GUANKE intake, which encourages its potential application as a probiotic in clinical trials.