In this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3'sialyllactose and 6'sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3'sialyllactose did not enhance adhesion. However, treatment with 6'sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3'- and 6'-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3'- and 6'-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6'sialyllactose, and 3'sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.