In colorectal cancer (CRC), macrophages represent a major component of the tumor mass and exert mostly functions promoting tumor cell survival, proliferation, and dissemination. Interleukin-34 (IL-34) is a cytokine overproduced by colon cancer (CRC) cells and supposed to make a valid contribution to the growth and diffusion of CRC cells. The biological functions of IL-34 are mediated by the macrophage colony-stimulating factor receptor (M-CSFR-1), which controls monocyte/macrophage differentiation, growth, and survival. We here investigated whether, in CRC, tumor-associated macrophages (TAMs) express M-CSFR-1 and functionally respond to IL-34. By flow-cytometry analysis of tumor-infiltrating cells (TICs) and lamina propria mononuclear cells (LPMCs) isolated from normal, adjacent mucosa of CRC patients, we showed that CD68/HLA-DRII-expressing TICs and LPMCs expressed M-CSFR-1. Both these cell types produced IL-34 even though the expression of the cytokine was more pronounced in TICs as compared to normal LPMCs. Moreover, in CRC samples, there was a positive correlation between IL-34-producing cells and CD68-positive cells. Stimulation of LPMCs and TICs with IL-34 resulted in enhanced expression of CD163 and CD206, two markers of type II-polarized macrophages, and this was evident at both RNA and protein level. In the same cell cultures, IL-34 stimulated expression and production of IL-6, a cytokine known to promote CRC cell growth and diffusion. Finally, knockdown of IL-34 in TICs with specific antisense oligonucleotides with: a specific antisesne oligonucleotide decreased IL-6 production and the number of TAMs producing this cytokine. This is the first to show a positive role of IL-34 in the control of TAMs in CRC, further supporting the notion that IL-34 sustains colon tumorigenesis.