10-Hydroxydec-2-enoic acid (10-HDA), the unique substance in the natural food royal jelly, is an unsaturated hydroxyl fatty acid with the bio-activity to guard against cell or tissue damages. However, the relevant mechanisms still remain largely unknown. Here, using a mouse erythrocyte model, whether the fluidity of plasma membrane is influenced by 10-HDA was investigated. 10-HDA was shown to enhance the erythrocyte membrane fluidity and to rescue the fluidity from •OH toxicity. In such occasions, 10-HDA promoted the dissolved O2 level in erythrocyte suspension thereof. The levels of β-actin and band 4.1 protein were not affected by 10-HDA in erythrocytes. In the framework of density functional theory, 10-HDA may interact with phosphatidylcholine and phosphatidylethanolamine, the major components of membrane lipid bilayer. This was evidenced by the fact that 10-HDA was detectable in erythrocytes after its addition to the erythrocyte suspension. Moreover, the fluorescent intensity of erythrocytes stained by the membrane probe DiIC18(5) increased with 10-HDA treatment. Hence, 10-HDA could regulate the erythrocyte membrane fluidity and the release of O2 from erythrocytes via targeting lipids rather than proteins. Our data cast light on the elucidation of the mechanism of 10-HDA modulating cell functionality and the utilization of 10-HDA in remedying the erythrocyte membrane fluidity-associated symptoms.