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Background: Diet is a modifiable risk factor for multiple cancers. It is also known to modulate gut microbial composition and metabolic activity, and plays an important role in maintaining gut homeostasis. However, our understanding of dietary quality and mucosa-associated microbiota in the large intestine in humans is limited. We examined the association between diet quality and gut microbiota in adults underwent routine colonoscopy in a cross-sectional study. Methods: We enrolled 21 men (50-75 years old, 71% White) who were found to have grossly normal colons at colonoscopy completed between July 2013 and April 2016. We obtained 98 colonic mucosa biopsies, with microbial DNA extracted from snap frozen biopsy. The 16S rRNA V4 region was amplified and sequenced on the Illumina MiSeq platform. The UPARSE and SILVA were used for operational taxonomic unit (OTU) classification. A self-administered BLOCK Food Frequency Questionnaire was used to assess dietary intake in the past year. Dietary quality was defined using the Healthy Eating Index (HEI)-2005, and further categorized as low or high using the median of total HEI or 12 individual component scores. We compared alpha-diversity (OTU and Shannon index), beta-diversity (Weighted UniFrac principal coordinates analysis), and relative abundance of bacterial phylum and genus by total HEI and all 12 individual HEI components using Mann-Whitney test. Reported P values were adjusted for multiple testing using false discovery rate. Results: The most abundant bacterial phyla observed were Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, and Fusobacteria. High-quality diet (total HEI score ≥ 63) was not associated with higher richness and evenness of gut microbiome (P = 0.12), but was significantly associated with bacterial composition (P = 0.046). Compared to men who had low-quality diet (total HEI score < 63), those with high-quality diet had a higher abundance of Proteobacteria (12.2% vs. 8.1%, P = 0.006), and lower abundance of Fusobacteria (0.13% vs. 3.37%, P = 0.004). At the genus level, those with high-quality diet had significantly lower abundance of Fusobacterium than those with low-quality diet (0.11% vs. 3.77%, P = 0.004). At the HEI component level, individuals with a higher consumption of solid fruits and milk had higher richness and evenness of gut microbiome (P < 0.05) as well as higher abundance of phylum Verrucomicrobia (P < 0.05) and genus Akkermansia (P < 0.001). Consuming more solid and saturated fat, alcohol, and added sugar were associated with a higher abundance of phylum Fusobacteria (P < 0.0001) and genus Fusobacterium (P = 0.0001). Conclusions: The colonic microbiome of men consuming a low-quality diet exhibited increased levels of Fusobacteria, which has been associated with colorectal cancer risk. If replicated in prospective research, our findings suggest low quality diet may contribute to colorectal cancer by modulating gut microbiome. Citation Format: Yanhong Liu, Nadim J. Ajami, Diane Hutchinson, David Graham, Sarah Plew, Ashley Johnson, Preksha Shah, Liang Chen, Kathryn Royse, Donna L. White, Jennifer Kramer, Matthew C. Wong, Rhonda Cole, Clark Hair, Jason Hou, Nisreen Husain, Maria Jarbrink-Sehgal, Fasiha Kanwal, Gyanprakash Ketwaroo, Rajesh Shah, Maria Velez, Melissa L. Bondy, Hashem B. El-Serag, Joseph F. Petrosino, Li Jiao. Healthy eating index 2005 and the mucosa associated gut microbiome in healthy individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3265.