Sesame protein isolate (SPI) as an encapsulation wall material was studied. The effect of microbial transglutaminase (MTGase) and also the encapsulation method (freeze drying (FD), spray drying (SD) and electrospraying (ES) methods) on the physicochemical characteristics and viability of Lactobacillus rhamnosus ATCC 7469 encapsulated in SPI was investigated. The hygroscopicity, moisture content, bulk density, aw, and color of microcapsules were affected by the encapsulation method. The particle size and zeta-potential of the microcapsules decreased and increased, respectively, after MTGase treatment. Irregular shapes, observed using field emission scanning electron microscope (FE-SEM), and the formation of new bonds, confirmed using Fourier transform infrared spectrometer (FT-IR), confirmed the successful encapsulation of L. rhamnosus using SPI. The SD method had the greatest protecting effect on L. rhamnosus with gastrointestinal conditions. Adding MTGase had a high protective effect on the viability of microcapsules at -18, 4, and 25 degrees C. Electrospraying had the poorest performance in the formation of SPI microcapsules. The SPI may be a potential candidate for the encapsulation of probiotics, and MTGase could improve the physical characteristics and microbial protection of the SPI-based microcapsules, especially using SD.