Spent mushroom substrate (SMS), a renewable bio-waste from the mushroom-growing industry, was used as an adsorbent to remove methylene blue (MB) from aqueous solution. SMS was characterized using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). Adsorption experiments with the SMS adsorbent were performed based on various parameters, such as adsorbent dose, initial MB dye concentration, initial pH, contact time, and temperature. The Langmuir, Freundlich, and Temkin isotherm models were employed to interpret the adsorption behavior. The results indicated that the equilibrium data were perfectly represented by the Temkin isotherm. The maximum adsorption capacity of SMS reached 63.5 mg g-1 at 303 K. The kinetics studies indicated that the pseudo-second-order model best described the adsorption of MB on SMS. The activation energy of the adsorption was 5.64 kJ mol-1. Thermodynamic parameters suggested that the adsorption was an exothermic and spontaneous physical process. The results imply that SMS is a potentially low-cost adsorbent for treating wastewater containing cationic dyes.