Objectives: Klebsiella oxytoca is a gastrointestinal pathobiont with the potential to produce the toxins tilivalline and tilimycin, which cause antibiotic-associated hemorrhagic colitis. Overgrowth of toxigenic K oxytoca has recently been implicated in necrotizing enterocolitis. K oxytoca colonizes 2-9% of healthy adults, however, there is no systematic data on colonization in healthy children. We investigated K oxytoca colonization and its toxigenic properties in healthy infants. Methods: We sampled stool of healthy infants and determined K oxytoca colonization using stool culture and PCR ( pehX). Toxin in stool was measured with HPLC/high-resolution mass spectrometry. K oxytoca isolates were typed using multi-locus sequence typing (MLST) and Koxytoca toxin PCR (npsA/B). Cytotoxin production of isolates was analyzed by MTT assay. Results: K oxytoca was detected in 30 of 61 infants (49%) using stool culture and in 45 of 61 (73%) using PCR ( pehX). Toxin marker PCR (npsA/B) was positive in 66% of stool samples positive for K oxytoca PCR. Stool toxin levels were too low for quantitation but traces of tilivalline were detected. Contrarily, 49% of K oxytoca isolates demonstrated toxicity in the MTT assay. MLST revealed 36 distinct sequence types affiliated with all known K oxytoca sequence type clusters (A, B1 and B2). Conclusions: More than 70% of healthy infants were colonized with K oxytoca. Toxin quantities in stool of colonized healthy infants were below detection level, yet half of the isolates produced toxin in vitro demonstrating their pathobiont potential. The high occurrence of toxigenic K oxytoca in healthy infants has to be considered for future disease association studies.