Hyaluronic acid (HA) is produced at extremely high levels in some breast cancer cells, which can then escape from apoptosis even if they become detached from the extracellular matrix. Activation of TRPV1, a non-specific cation channel protein, has been shown to cause apoptosis in these cells. Therefore, using MCF-7 cells, we investigated the expression of the HA receptor CD44 and TRPV1 when the cells were placed on a low-adhesive scaffold. Then, we examined the effects of the HA-synthesis inhibitor 4-methylumbelliferone (4-MU) on TRPV1 expression as well as the effect of TRPV1 modulators on 4-MU–induced apoptosis. We also tested the direct action of HA on TRPV1 activity by using a Ca imaging system. In cancer spheroids formed on the poly (2-hydroxyethylmethacrylate)-coated dishes, both HA production and CD44 mRNA expression were increased, whereas TRPV1 mRNA expression was decreased. 4-MU inhibited both HA production and CD44 expression but increased TRPV1 mRNA expression and protein production. The TRPV1 agonist capsaicin increased the amount of Annexin V/PI-positive cells, the action of which was inhibited by the TRPV1 antagonist AMG9810. Furthermore, 4-MU–induced apoptosis was strongly suppressed by AMG9810. HA itself inhibited the capsaicin-induced increase in Ca²⁺ in TRPV1-transfected HEK293 cells. These results suggest that during cell detachment, the HA-CD44 pathway is likely to be involved in the underlying mechanism of apoptosis avoidance by inhibiting the expression and function of TRPV1.