In this study, adsorption/desorption of vancomycin (VAN) on bentonite nanoparticles was investigated in a batch system. Adsorption experiments were carried out as a function of several influential parameters such as adsorbent dosage, pH, contact time and ionic strength. Bentonite nanoparticles were characterized by field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, Brunauer–Emmett–Teller, and Fourier transform infrared (FTIR) analyses and the mesoporous structure was revealed. Langmuir, Freundlich, and Temkin isotherm models were applied for the examination of equilibrium data, and Langmuir was found to be the best fit. With the increase in pH and ionic strength, the adsorption capacity decreases, which suggests the adsorption process may be dominated by the cation exchange mechanism. Moreover, VAN desorption from bentonite nanoparticles in two initial VAN loadings was investigated under different concentrations of metallic cations of various valences (Na+, Ca2+, Al3+), and pHs 3–10. Desorption was strongly pH-dependent and the amount of VAN desorbed increased with increasing cations concentrations. The FTIR analysis before and after VAN desorption suggests that the formation of Al-VAN and Ca-VAN complexes on the solid surface and then their detachment from the solid surface may contribute to the higher VAN desorption by Al3+ and Ca2+.