Hyaluronan (HA), through interaction with its receptor CD44, induces tumor progression. ErbB-2, a member of ErbB family of membrane receptor tyrosine kinases, migrates to the nucleus (NErbB2) where it acts as a transcription factor/coactivator to modulate proliferation and resistance to anti-ErbB-2 agents in breast cancer (BC). Accumulation of HA is associated with poor prognosis and promotes resistance to anti-ErbB-2 agent trastuzumab (TZ) in BC. Although crosstalk between HA/CD44 and ErbB-2 pathways has been reported, how their molecular interactions mediate TZ resistance remains unknown. Our in silico studies showed that TZ-resistant cells presented higher CD44 levels than TZ-sensitive ones. Stimulation with the ErbBs ligand heregulin (HRG) induces NErbB-2 translocation, acquired-TZ resistance and proliferation in SKBR3 cells. HRG also increased CD44 expression in SKBR3. In a de novo TZ-resistant BC model, JIMT1, the constitutive levels of nuclear CD44 (NCD44) and NErbB-2 were further enhanced by HA stimuli. Treatment with the chemical inhibitor of HA synthesis 4-methylumbelliferone (4MU) decreased not only HA levels but also NErbB-2 in JIMT1 cells. Furthermore, 4MU inhibited proliferation and migration of JIMT1 cells similarly to the inhibition observed when ErbB-2 was excluded from the nucleus via transfection with hErbB-2ΔNLS mutant. 4MU also inhibited HRG-induced proliferation in SKBR3. Our findings highlight the blockade of HA synthesis as a novel therapeutic strategy in TZ-resistant BC.