Hyaluronan (HA), a polymer with various molecular weights (MW) found in tumor microenvironments, is associated with malignant progression of breast cancer. Reducing the amount of high-MW HA in the microenvironment by hyaluronidase is a promising approach for breast cancer treatment. However, whether the generation of HA fragments negatively affects breast cancer cells remains to be determined. Furthermore, HA forms three-dimensional (3D) networks by cross-linking with other extracellular molecules to function. Therefore, a model mimicking the cross-linked HA network is required to determine the effect of HA fragments on breast cancer cells. To clarify the differential roles of low (HA35) versus high (HA117) MW HA on cancer cell phenotype, a 3D culture system was set up by covalently cross-linking HA with alginate and investigating the behavior of 4T-1 and SKBR3 breast cancer cells alongside a two-dimensional (2D) control. The results show the invasion and migration abilities of 4T-1 and SKBR3 cells are significantly enhanced by the presence of HA35 but inhibited by HA117 in both 2D monolayers and 3D spheroids. The differential effects of HA35 and HA117 on cancer cell epithelial-mesenchymal transition (EMT) phenotype were further confirmed in terms of differential regulation of E-cadherin and vimentin as important EMT markers at both the cellular and mRNA levels. Additional experiments show the CD44-Twist signaling pathway might be involved in the differential effects of HA35 and HA117. These results have important implications with respect to understanding the role of HA in breast cancer development and for the design of therapeutic approaches based on the eradication of HA with hyaluronidase.