An inflammatory microenvironment is a tumour-promoting condition that provides survival signals to which cancer cells respond with gene expression changes. One example is the alternative splicing variant Rat Sarcoma Viral Oncogene Homolog (Ras)-Related C3 Botulinum Toxin Substrate 1 (RAC1)B, which we previously identified in a subset of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-mutated colorectal tumours. RAC1B was also increased in samples from inflammatory bowel disease patients or in an acute colitis mouse model. Here, we used an epithelial-like layer of polarized Caco-2 or T84 colorectal cancer (CRC) cells in co-culture with fibroblasts, monocytes or macrophages and analysed the effect on RAC1B expression in the CRC cells by RT-PCR, Western blot and confocal fluorescence microscopy. We found that the presence of cancer-associated fibroblasts and M1 macrophages induced the most significant increase in RAC1B levels in the polarized CRC cells, accompanied by a progressive loss of epithelial organization. Under these conditions, we identified interleukin (IL)-6 as the main trigger for the increase in RAC1B levels, associated with Signal Transducer and Activator of Transcription (STAT)3 activation. IL-6 neutralization by a specific antibody abrogated both RAC1B overexpression and STAT3 phosphorylation in polarized CRC cells. Our data identify that pro-inflammatory extracellular signals from stromal cells can trigger the overexpression of tumour-related RAC1B in polarized CRC cells. The results will help to understand the tumour-promoting effect of inflammation and identify novel therapeutic strategies.