Lactic acid bacteria (LAB) with probiotic properties are useful options for prophylactic and therapeutic applications against gastrointestinal diseases. The safety of probiotics should, however, be verified before incorporation into food or drinks. The present study had encapsulated Lactobacillus plantarum LAB12 within microcapsules that could withstand extremely high temperature (up to 100 °C) during pelletisation. The microencapsulated LAB12 were then tested for their acute (single dosing) and sub-chronic (a 90-day feeding) toxicity. For acute toxicity study, six male Sprague-Dawley rats were being administered with a single dose of freeze-dried microencapsulated LAB12 at 11 log CFU/kg BW through oral gavage. No clear treatment-related effects were observed after 14 days. For sub-chronic toxicity study, rodents were randomly divided into four groups (6 rats/sex/group) and treated with 0, 8, 9 and 10 log CFU/kg BW of microencapsulated LAB12 in pellet form. No mortality or treatment-related findings were observed in terms of clinical body weight, water intake, or food consumption. No treatment-related adverse effects were observed in blood and tissue samples. The no-observed-adverse-effect-level (NOAEL) for microencapsulated LAB12 was 2.5 × 1010 CFU/kg BW for both genders. These results imply that LAB12 are likely non-pathogenic and non-toxic.