Abstract: For further understanding the wettability alteration induced by organic salts, series of bis-imidazolium salts (EBMI, TBMI, HBMI, OBMI and DBMI) were employed for investigating their adsorption behavior and wettability alteration on vermiculite (Vt) by experimental and theoretical studies. The characterization results indicated that all bis-imidazolium salts had been loaded on Vts. The adsorption results showed that EBMI, TBMI, HBMI, OBMI and DBMI on Vt reached equilibrium of 0.159, 0.156, 0.145, 0.114 and 0.084 mmol g−1 around 30 min at 25 °C, respectively, which were sensitive to ionic strength and pH. Langmuir, statistical physical modelling and pseudo-second-order models could be well fitted with the adsorption data, and thermodynamic parameters suggested that the adsorption processes of bis-imidazolium salts were endothermic and spontaneous, indicating that the resultant bis-imidazolium salts could be self-assembled onto Vt in the form of the monolayer. Results of molecular dynamic simulation showed that bis-imidazolium salts were adsorbed on Vt with the lying-flat configuration, and the electrostatic interaction acted as the main interaction mechanism, which were consistent with that obtained experimentally. Changes of wettability of Vt induced by bis-imidazolium salts were verified by capillary rise experiments. Interestingly, the wettability of organo-Vts varied with the spacer length and the order was as follows: EBMI-Vt < TBMI-Vt < HBMI-Vt < OBMI-Vt < DBMI-Vt, which could be explained by their arrangements, hydrophobicity as well as the interaction energies. The longer the spacers of bis-imidazolium salts, the greater the absolute values of the interaction energy, the less the adsorbed bis-imidazolium salts, while the more hydrophobic of organo-Vt. This work aimed at revealing the adsorption behavior, mechanism as well as effect of bis-imidazolium salts on wettability alteration of negatively charged mineral surface, providing some information for the selection of flooding agent for enhanced oil recovery and wettability modifier.