Recently, a paste-type premixed calcium silicate-based mineral trioxide aggregate (MTA) product that quickly solidifies through a pozzolanic reaction was introduced to replace existing MTA, which has the disadvantage of a long setting time. In this study, we evaluated the effect of moisture content in the root canal on the setting time of premixed calcium silicate-based MTA in a simulated root canal environment using Endoseal MTA and Well-Root ST, among commercially available products. The setting time was measured according to ISO 6876/2012. A mold made using grades 2, 3, and 4 dental gypsum according to the classification of ISO 6873/2013 was used to reproduce the difference in moisture environment. Differences in moisture content were measured using micro-computed X-ray tomography (micro-CT). The micro-CT results showed that the moisture content was the highest and lowest in the grade 2 and 4 gypsum molds, respectively. Moreover, the setting time indicated by the manufacturer was the shortest for the grade 2 gypsum mold. Hence, the differences in moisture content significantly affect the setting time of MTA. This result can help set future experimental conditions and develop premixed calcium silicate-based MTA products.