Background: The incidence of colorectal cancer (CRC) in young adults ( < 50 years old) has been rapidly increasing by 2% per year since early 1990. Approximately 20% of early-onset (EO) CRC cases are due to germline gene mutations. However, the etiology of sporadic EO CRC remains poorly understood. Research suggests that environmental factors such as the Western diet (high in fat and low in fiber) may be associated with an increased incidence of sporadic EO CRC. The gut microbiota decompose and ferment dietary fibers to produce microbial metabolites, which play pivotal roles in maintaining the integrity of intestinal epithelium as well as the immune cell homeostasis. Also, these microbial metabolites may influence the host epigenome by altering either the activity of epigenetic enzymes or by modifying the availability of cofactors needed for epigenetic modifications. The aim of our research is to associate intratumoral microbiota with methylation pattern and immune cell composition in EO CRC. Methods: A total of 358 CRC cases, including 54 cases of EO CRC (age < 50 years) and 304 cases of late onset (LO) CRC (age ≥ 50 years), with matched methylation array (Infinium HM450), RNA-sequencing (Illumina HiSeq) from colon adenocarcinomas (COAD) and rectal adenocarcinomas (READ), and clinicopathological information of each patient, were extracted from the Cancer Genome Atlas (TCGA). We characterized and compared the intra-tumoral microbiota composition, tumor-infiltrating lymphocytes (TILs), and methylation profile between EO and LO CRC. Results: We found that there is a distinct microbial distribution, gene expression and methylation pattern in the EO CRC when compared with LO CRC. Non-human sequences from several kingdoms including bacteria, fungi and viruses were found and the incidences were consistent with reported values by other methods, e.g. Fusobacterium incidence. The EO CRC cases showed global hypomethylation, even though hypermethylation pattern is expected in the young chronological age group (known as Horvath’s clock). Pathway overrepresentation analysis of differentially expressed genes showed significant activation of p53 and pentose phosphate pathways and de novo nucleotide synthesis in EO CRC. Integration across datasets showed positive correlations between microbes and inflammasome pathway, positive correlation with regulatory T cells (Tregs), and negative correlations with CD4 memory T cells. Conclusions: These data suggest a mechanism by which the colorectal cancer-associated microbiota may be associated with epigenetic regulation and host immune response.