Abstract Chronic infection and associated inflammation have long been suspected to promote human carcinogenesis. Recently, certain gut bacteria, including some in the Fusobacterium genus, have been implicated in playing a role in human colorectal cancer (CRC) development. However, the Fusobacterium species and subspecies involved and their oncogenic mechanisms remain to be determined. We sought to identify the specific Fusobacterium spp. and ssp. in clinical CRC specimens by targeted sequencing of Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium spp. were identified in clinical CRC specimens. Additional analyses confirmed that Fusobacterium nucleatum ssp. animalis was the most prevalent F. nucleatum subspecies in human CRCs. We also assessed inflammatory cytokines in CRC specimens using immunoassays and found that expression of the cytokines interleukin-17A and tumor necrosis factor-alpha was markedly increased but interleukin-21 decreased in the colorectal tumors. Furthermore, the chemokine (C-C motif) ligand 20 was differentially expressed in colorectal tumors at all stages. In in vitro co-culture assays, F. nucleatum ssp. animalis induced CCL20 expression in CRC cells and monocytes. It also stimulated the monocyte/macrophage activation and migration. Our observations suggested that infection with F. nucleatum ssp. animalis in colorectal tissue could induce inflammatory response and promote CRC development. Further studies are warranted to determine if F. nucleatum ssp. animalis could be a novel target for CRC prevention and treatment. Citation Format: Xiangcang Ye, Rui Wang, Rajat Bhattacharya, Delphine R. Boulbes, Fan Fan, Ling Xia, Adoni Harish, Nadim J. Ajami, Matthew C. Wong, Daniel P. Smith, Joseph F. Petrosino, Susan Venable, Wei Qiao, Veera Baladandayuthapani, Dipen Maru, Lee M. Ellis. Fusobacterium nucleatum subspecies animalis influences pro-inflammatory cytokine expression and monocyte activation in human colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2674. doi:10.1158/1538-7445.AM2017-2674