New treatment targets are needed for colorectal cancer (CRC). We define expression of High Mobility Group Box 1 (HMGB1) protein throughout colorectal neoplastic progression and examine the biological consequences of aberrant expression. HMGB1 is a ubiquitously expressed nuclear protein that shuttles to the cytoplasm under cellular stress. HMGB1 impacts cellular responses, acting as a cytokine when secreted. A total of 846 human tissue samples were retrieved; 6242 immunohistochemically stained sections were reviewed. Subcellular epithelial HMGB1 expression was assessed in a CRC Tissue Microarray ( n = 650), normal colonic epithelium ( n = 75), adenomatous polyps ( n = 52), and CRC polyps (CaP, n = 69). Stromal lymphocyte phenotype was assessed in the CRC microarray and a subgroup of CaP. Normal colonic epithelium has strong nuclear and absent cytoplasmic HMGB1. With progression to CRC, there is an emergence of strong cytoplasmic HMGB1 ( p < 0.001), pronounced at the leading cancer edge within CaP ( p < 0.001), and reduction in nuclear HMGB1 ( p < 0.001). In CRC, absent nuclear HMGB1 is associated with mismatch repair proteins ( p = 0.001). Stronger cytoplasmic HMGB1 is associated with lymph node positivity ( p < 0.001) and male sex ( p = 0.009). Stronger nuclear ( p = 0.011) and cytoplasmic ( p = 0.002) HMGB1 is associated with greater CD4 + T-cell density, stronger nuclear HMGB1 is associated with greater FOXP3 + ( p < 0.001) and ICOS + ( p = 0.018) lymphocyte density, and stronger nuclear HMGB1 is associated with reduced CD8 + T-cell density ( p = 0.022). HMGB1 does not directly impact survival but is associated with an 'immune cold' tumour microenvironment which is associated with poor survival ( p < 0.001). HMGB1 may represent a new treatment target for CRC.