Ethidium bromide (EtBr)-containing wastewater can be hazardous to biodiversity when released into the soil and water bodies without treatment. EtBr can mutate living microbial cells and pose toxicity to even higher organisms. This work investigated the removal of EtBr from aqueous solutions by a naturally occurring palygorskite (PFl-1) clay mineral via systematic batch adsorption experiments under different physicochemical conditions. EtBr existed in an undissociated form at pH ~7, and was adsorbed on PFl-1 obeying the Freundlich isotherm model. The maximum EtBr adsorption capacity was 285 mmol/kg. The best fitted kinetic model for EtBr adsorption was the pseudo-second order model. The amounts of exchangeable cations desorbed from PFl-1 during EtBr adsorption was linearly correlated to the amounts of EtBr adsorbed, with a slope of 0.97, implying that a cation exchange-based adsorption mechanism was dominating. Additionally, dimerization of EtBr molecules via bromide release assisted an increased EtBr removal by PFl-1 at high adsorbate concentrations. Detailed x-ray diffraction, Fourier transform infrared, scanning electron imaging and energy dispersive x-ray analyses confirmed that EtBr adsorption occurred dominantly on the surface of palygorskite which mineralogically constituted 80% of the bulk PFl-1 adsorbent. A small portion of EtBr was also adsorbed by PFl-1 through intercalation onto the smectite impurity (10%) in PFl-1. This study suggested that PFl-1 could be an excellent natural material for removing EtBr from pharmaceutical and laboratory wastewater.