Background: Bee pollen (BP) has a broad range of beneficial effects on health. The aim of this study was to examine the effect of BP on the oral environment, including the microbiome and antimicrobial peptides. Methods: C57BL/6J mice were randomly divided into two groups: control and BP. The BP group was fed with a 5% BP diet for 1 month. Swabs from the oral and buccal mucosa and samples of the intestinal stool were collected. Genomic DNA was extracted and the microbiome was analyzed via 16S rRNA sequencing. Results: BP inhibited the growth of P. gingivalis at a concentration of >2.5%. The metagenomic study showed that the abundance of genus Lactococcus was significantly elevated in the oral and intestinal microbiomes of the BP group when compared to those of the control group. Significant alterations in alpha and beta diversity were observed between the oral microbiomes of the two groups. The mRNA levels of beta-defensin-2 and -3 were significantly upregulated in the buccal mucosa of the BP group. Conclusion: A BP diet may have a beneficial effect on oral and systemic health by modulating the bacterial flora and antimicrobial peptides of the oral cavity. Further investigations are needed to clarify how a BP diet affects overall human health.