Hyaluronan (HA) cross-linking is a conformational state of HA, a covalent complex between HA and heavy chains (HCs) from inter-α-trypsin inhibitor (I-α-I) mediated by tumor necrosis factor-induced protein 6 (TSG6). Cross-linked HA has been identified as a protective factor in physiological and inflammatory conditions. However, the state of HA cross-linking in tumor microenvironment has not been fully elucidated. As a major constituent of the extracellular matrix (ECM), HA is mainly synthesized by cancer-associated fibroblasts (CAFs). Our study aimed to clarify the role of HA cross-linking in breast cancer malignancy. Compared to normal mammary gland tissues, cross-linked HA levels were significantly decreased in breast cancer and associated with tumor malignancy. When NFbs were activated into CAFs, the levels of cross-linked HA and TSG6 were both suppressed. Through upregulating TSG6, CAFs restored the high level of cross-linked HA and significantly inhibited breast cancer malignancy, whereas NFbs promoted the malignancy when the cross-linked HA level was reduced. Furthermore, the inhibitory role of HA cross-linking in tumor malignancy was directly verified using the synthesized HA-HC complex. Collectively, our study found that the deficiency of cross-linked HA induced breast cancer malignancy in a CAF-dependent manner, suggesting that recovering HA cross-linking may be a potential therapeutic strategy.