Objective To investigate the effect of adhesion behavior of the extraocular muscle ( EOM) on eye motion. Methods The pull-off test was carried out on EOM in vitro exposed to the air in different time periods using steel spherical indenters with three different sizes, and adhesion behavior of the EOM was studied. Then contribution of the adhesion between EOM and sclera to maintain eye balance during eye motion was analyzed by combination with the JKR theory. Results The adhesion force of the epimysium of EOM in vitro decreased with the increase of exposure time. Except for the case of 1-hour exposure time, the adhesion force was not affected by the indenter size at other exposure time. The experimental adhesion force was in good agreement with the theoretical adhesion force calculated by the JKR theory. The adhesion force between EOM and sclera, as well as the active force of lateral rectus muscle increased with the abduction angle increasing. The adhesion moment increased exponentially with the abduction angle, and accounted for an increasing proportion of the sum of resistance moment, passive moment and adhesion moment. Conclusions The duration of exposure to the air had a significant effect on adhesion properties of the epimysium of EOM in vitro. The adhesion interaction between sclera and EOM could be preferably described by the JKR theory. The adhesion interaction between EOM and sclera participated in maintaining balance of the eyeball and made certain contributions in regulating eye motion. The results in this study can provide theoretical foundation for researches of disorders related to eye motion such as strabismus.