Lactobacillus reuteri NCIMB 30253 was shown to have potential as a probiotic by reducing the proinflammatory chemokine interleukin-8. Moreover, this strain was evaluated, by in vitro and in vivo techniques, for its safety for human consumption. The identity of the strain was investigated by metabolic profiling and 16S rRNA gene sequencing, and in vitro safety evaluations were performed by molecular and metabolic techniques. Genetic analysis was confirmed by assessing the minimum inhibitory concentration to a panel of antibiotics, showing that the strain was susceptible to 8 antibiotics tested. The ability of the strain to produce potentially harmful by-products and antimicrobial compounds was evaluated, showing that the strain does not produce biogenic amines and does not show bacteriocin activity or reuterin production. A 28-day repeated oral dose study was conducted in normal Sprague-Dawley rats to support the in vivo strain safety. Oral administration of the strain resulted in no changes in general condition and no clinically significant changes to biochemical and haematological markers of safety relative to vehicle control treated animals. This comprehensive assessment of safety of L. reuteri NCIMB 30253 supports the safety of the strain for use as a probiotic.