Nanoclays have been developed as drug delivery systems, but their mechanisms of DOX delivery are unclear. Herein, unmodified nanoclays (halloysite, kaolinite, montmorillonite) were comprehensively studied on their in vitro binding and release mechanisms of DOX from both experimental and theoretical aspects. These nanoclays with high loading capacity (>50%) and encapsulation efficiency capacity (>90%) of DOX are attributed to the exposed hydroxyl groups and the Lewis base sites on the surfaces. Density functional theory calculations also confirmed that DOX is preferentially adsorbed on the Al-OH surfaces while adsorption on Si-O surfaces is limited. Besides this, the pH-responsive profiles of DOX release from nanoclays are related to the protonation of negatively charged nanoclays in weakly acidic solutions that makes it easier to dissociate with positively charged DOX. The in-depth mechanistic method in this work is widely applicable and demonstrates that nanoclays can be used as efficient nanocarriers for more biomedical applications.