In Europe, the species Lactobacillus rhamnosus is currently on the Qualified Presumption of Safety list used by the European Food Safety Authority (EFSA) for internal safety assessment, but according to the EFSA the species should remain a topic of surveillance. In the present study, the safety and tolerance of the potentially probiotic strain L. rhamnosus PRSF-L477 was investigated in a placebo-controlled double-blind volunteer trial following FAO/WHO guidelines. A total of thirty-four subjects received daily doses of 1 × 1011 colony-forming units (cfu) of L. rhamnosus PRSF-L477 (n 17) or placebo (n 17) for a period of 3 weeks, followed by a wash-out period of another 3 weeks. A questionnaire on gastrointestinal tolerance and a diary was kept daily to record compliance throughout these 6 weeks. Faecal and blood samples were collected for microbiological and haematological analysis. The recorded gastrointestinal symptoms, defecation frequency and stool consistency were not influenced indicating that L. rhamnosus PRSF-L477 was well tolerated. The species L. rhamnosus was detected in the faeces of sixteen out of seventeen subjects of the probiotic group during the intervention period. Using pulsed-field gel electrophoresis, re-isolates of L. rhamnosus PRSF-L477 were confirmed in nine of these subjects. Antibiotic susceptibility profiles of these re-isolates were unchanged compared with PRSF-L477. No clinically relevant changes in blood parameters such as liver and kidney function and no serious adverse events appeared during and after administration. Therefore, we conclude that L. rhamnosus PRSF-L477 can safely be administrated to healthy subjects at a daily dose of 1 × 1011 cfu.