Background: Toll-like receptors (TLRs) are involved in innate immunity. Overexpression of TLRs has been implicated in various types of cancer including colorectal cancer (CRC). The phosphatidylinositol-3'-kinase (PI3K)/Akt signaling pathway is involved in CRC growth and progression. In this study, we determined whether TLR4 signaling and PI3K/Akt pathway activation occur in CRCs. Materials and Methods: Human CRCs and adjacent mucosa were evaluated for TLR4 expression. CRC cell lines were treated with lipopolysaccharide (LPS), endogenous TLR4 ligand, to assess Akt phosphorylation. Results: Human CRCs overexpressed TLR4 compared to matched normal mucosa. Additionally, TLR4 was expressed in CRC cells and LPS treatment increased Akt phosphorylation of TLR4-positive CRCs in a time-dependent manner. Conclusion: Our results identify TLR4 expression in human CRCs and activation of PI3K with LPS treatment. These findings suggest possible treatment strategies targeting TLR4 in CRC.