Background: Transforming growth factor- β pathway (TGF-β) has an established role in promoting growth, invasion, metastasis as well as epithelial to mesenchymal (EMT) transition. Among 4 different described molecular subtypes of colorectal cancer (CRC), consensus molecular subtype 4 (CMS4) comprises up to 25% of CRC pts, distinguished by activation of this pathway, and is associated with higher relapse rate and poor prognosis. Recently, it has also been proposed that TGF-β activation drives immune evasion in murine models, but these findings have not been clinically validated. Methods: Using multi-gene RNA expression profiling, fresh-frozen paraffin-embedded samples of 35 patients with CRC were analyzed to determine TGF-β and EMT expression levels. Multiplexed IHC staining was performed on FFPE tumor blocks by using the Opal 7-Color fIHC Kit and the stained slides were scanned by a Vectra multispectral microscope (PerkinElmer) to measure infiltration of immune cells (i.e., T lymphocytes, cytotoxic T lymphocytes (CTL), T cell antigen-experienced, macrophages, etc.) in the tumor, stroma, and both components. TGF-β and EMT expression levels – as continuous variables - were compared with the infiltration of various immune cells using Spearman’s rank correlation analysis. Results: Among 35 pts, 28 pts had non-CMS1/MSS CRC. TGF-β RNA expression in the tumor microenvironment of these samples was inversely associated with the infiltration of CTL into the tumor (r=-0.43, p= 0.022). In contrast, there was no association of TGF-β with non-cytotoxic T-cells or macrophage infiltration. The tumor and stromal CTL infiltration differed substantially by CMS (p=0.04, p=0.02, respectively) with tumor infiltration lowest in CMS4 (n=7). Consistent with this, EMT gene signature, which includes TGF-β expression, showed a similar inverse correlation with CTL infiltration (r=-0.48, p=0.009). Conclusions: TGF-β and EMT gene signatures have important roles in the exclusion of CTL in the tumor microenvironment of CRC pts. Inhibiting TGF-β pathway can potentially increase the intratumoral infiltration of CTL, which is a necessary (but not sufficient) step for immunotherapy response in MSS CRC. Clinical trials evaluating this hypothesis are currently ongoing (NCT03436563).