The tumor microenvironment (TME), consisting of stromal fibroblasts, immune cells, cancer cells and other cell types, plays a crucial role in cancer progression and metastasis. M2 macrophages and activated fibroblasts (AFs) modulate behavior of cancer cells in the TME. Since nutritional effects on cancer progression, including colorectal cancer (CRC), may be mediated by alterations in the TME, we determined the ability of β-carotene (BC) to mediate anti-cancer effects through regulation of macrophage polarization and fibroblast activation in CRC. The M2 macrophage phenotype was induced by treating U937 cells with phorbol-12-myristate-13-acetate and interleukin (IL)-4. Treatment of these M2 macrophages with BC led to suppression of M2-type macrophage-associated markers and of the IL-6/STAT3 signaling pathway. In separate experiments, AFs were induced by treating CCD-18Co cells with transforming growth factor-β1. BC treatment suppressed expression of fibroblast activation markers. In addition, conditioned media from BC-treated M2 macrophages and AF inhibited cancer stem cell markers, colon cancer cell invasiveness and migration, and the epithelial-mesenchymal transition (EMT). In vivo, BC supplementation inhibited tumor formation and the expression of M2 macrophage markers in an azoxymethane/dextran sodium sulfate-induced colitis-associated CRC mouse model. To our knowledge, the present findings provide the first evidence suggesting that the potential therapeutic effects of BC on CRC are mediated by the inhibition of M2 macrophage polarization and fibroblast activation.Copyright © 2020. Published by Elsevier Inc.