This article investigates the impact of sesame protein isolate (SPI) and increased calcium in milk on the biochemistry, rheology and micro-structure of cheese. The characteristics of two sets of cheeses made from raw milk and milk of 8% protein substituted by SPI (SPI milk) with 0, 5, 10 and 20 mM calcium addition were investigated. Adding SPI to milk resulted in rennet gel formation with longer coagulation time and lower storage modulus (G′) at 60 min in cheesemaking. The synergistic influence of SPI and calcium resulted in increased hardness, cohesiveness, adhesiveness and meltability during 21 days of cheese aging. Adding calcium to SPI milk resulted in a more compact and regular micro-structure in cheese. Scanning electron microscopy indicated the associative effect of sesame protein and calcium generated greater aggregation in complex cheese micelles, which was key in SPI cheese property formation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel electrophoretograms of the wheys and caseins indicated the interaction of sesame protein and casein with calcium. This study is of benefit in the manufacture of novel cheese with traditional orient food to meet with the taste of Chinese people.