Background: Agaro-oligosaccharides (AOS) have been shown to modulate the gut microbiota in in vitro and animal studies; however, human evidence remains scarce. Methods: Herein, we conducted a four-week open-label, single-arm, non-randomized pilot trial in 18 healthy Japanese adults to examine the association of AOS intake at 200 mg/day with gut microbiota composition and bowel condition. Fecal samples collected before and after the intervention were analyzed using QIIME2-based 16S rRNA sequencing, and bowel condition was assessed with the Bristol Stool Form Scale. This study was registered in the UMIN Clinical Trials Registry (UMIN000056992). Results: AOS intake was not associated with significant changes in bowel condition. Gut microbiota analysis showed no significant alterations in overall community structure but revealed taxon-specific trends in the relative abundance of several bacterial taxa. Notably, nominal changes were observed in the abundance of the Ruminococcus gnavus group and Bacteroides uniformis after the intervention. In addition, quantitative PCR analysis showed an increase in 3,6-anhydro-L-galactose cycloisomerase (ACI) gene abundance after the intervention. Conclusions: These findings suggest that, in this exploratory pilot study, AOS intake was associated with a taxon-specific pattern in the gut microbiota. Further randomized controlled studies are needed to clarify the microbiota-related effects of AOS in humans.