This study conducted a detailed investigation on the influence of the culture medium type on the permeability reduction effect through microbial-induced calcium carbonate precipitation (MICP) technology in a single rough fracture. The differences between the Sporosarcina pasteurii cultured by two mediums were compared in terms of bacterial growth properties, permeability treatment effects, distribution characteristics of induced CaCO3, and microscopic crystal characteristics. The study revealed that the culture medium did indeed impact the permeability reduction effect when treated with MICP technology, primarily related to whether urea is added in the culture medium. Two distinct permeability reduction modes were proposed for the treatment process using Sporosarcina pasteurii cultured by different mediums through one-phase injection (mixing bacterial solution and cementing solution). The permeability decreased rapidly, and the distribution of induced CaCO3 was uneven after treatment with Sporosarcina pasteurii cultured in medium containing urea, while the permeability decreased relatively slowly and the induced CaCO3 distribution was relatively uniform when treated with Sporosarcina pasteurii cultured in medium without urea. Additionally, differences in crystal morphology were observed due to variations in seepage characteristics during the treatment process with the Sporosarcina pasteurii cultured by different mediums. Finally, some investigations were given to the treatment effect optimization for the treatment process of Sporosarcina pasteurii cultured in medium containing urea. This study gave an insight to the regulation mechanism of culture medium and treatment method for the process of permeability reduction by MICP technology in the rock fracture.