Metabolism, Organ Distribution, and Blood-Brain Barrier Penetration of Human Milk Oligosaccharides—6′/3′-Sialyl-N-acetyllactosamine

3′-sialyl-N-acetyllactosamine (3′-SLN) and 6′-sialyl-N-acetyllactosamine (6′-SLN) are two important human milk oligosaccharides (HMOs) which play significant functions in brain development and antiviral potential. However, their metabolism is still unknown. In this study, chemoenzymatically synthesized 3′-SLN and 6′-SLN were labeled with cyanine-7 (Cy7) via formation triazole (Tz) derivatives to investigate their metabolism and organ distribution in a mouse model. The fluorescence signals were detected in the brains of mice after 0.5 h of gavage with 3′-SLN-Tz-Cy7 and 6′-SLN-Tz-Cy7. It was found for the first time that both of them can cross the blood–brain barrier (BBB) as a whole and reach the brain in a sex-specific manner. And the results show that, whether in the in vivo imaging results or the brain fluorescence signal results, the male mice absorbed 3′-SLN-Tz-Cy7 more than 6′-SLN-Tz-Cy7; meanwhile, in the female mice, the results were exactly the opposite. Both 3′-SLN-Tz-Cy7 and 6′-SLN-Tz-Cy7 exhibited the highest fluorescence intensity in pulmonary tissues, followed by substantial hepatic deposition. This study would offer preliminary evidence for the hypothesis that the oral administration of 3′-SLN and 6′-SLN may promote brain development and provide a foundation for the further exploration of their functions in brain cognition.