Hyaluronic acid (HA) is found in various tumor tissues, and is considered tumor-associated extracellular matrix (ECM). Within this tumor-associated ECM, stromal cells, especially immune cells, are involved in tumor progression. However, the effects of tumor-associated ECM on the characteristics of immune cells remain unexplored. Therefore, we studied the triggering effect of HA on spontaneous M2-like polarity of monocytes/macrophages using HA-mixed collagen (HA-COL) matrix. In the presence of HA, expression of the HA receptor (CD44) and M2 polarity-related genes was upregulated in human monocytes (THP-1 cells). We confirmed the CD44-mediated activation of STAT3 in THP-1 cells cultured in an HA-rich environment. Furthermore, when we induced the THP-1 cells to differentiate into cells with M1 or M2 polarity within an HA-rich environment, the HA-rich environment influenced the direction of induction. Our findings might improve understanding of the crosstalk between immune cells and tumor-associated ECM, and facilitate development of tumor immunotherapy strategies.