The chymosin-induced coagulation of microfluidized milk was evaluated to determine its potential in making high-moisture cheese. Raw, thermized (65C, 15s) and HTST pasteurized (75C, 15s) 3.0% (wt/wt) fat milk samples were microfluidized at 4 treatment conditions: 42C/75 MPa, 42C/125 MPa, 54C/125 MPa, and 54C/170 MPa; controls consisted of non-homogenized and homogenized (2-stages at 10/5 MPa) milk. Microfluidized and non-microfluidized milk samples were evaluated for alkaline phosphatase activity and median particle size. After chymosin addition, coagulation time, curd firmness and microstructure were examined. Microfluidization had varied and significant effects (P < 0.05) on the size reduction of fat droplets, coagulation time, and curd firmness, while either heat treatment alone did not. Alkaline phosphatase was inactivated in all samples except for the raw and thermized milk non-microfluidized controls and the 75 MPa treatment. Particle size decreased by half during 2-stage homogenization, and 15- to 20-fold after microfluidization yielding median area-weighted values, d(3,2), of 7.9, 4.2, and 0.39 to 0.50 μm, respectively. Milk samples microfluidized at 42C and 75 or 125 MPa were similar to the controls in coagulation times and curd firmness. Compared with the controls, milk microfluidized at 54C and 125 or 170 MPa took 3 to 8 times longer to coagulate and had lower curd firmness, which indicated that protein matrix formation, a critical step in the production of cheese, had been altered. Scanning electron microscopy images of the chymosin curds illustrated that, compared with the controls, the use of nonthermal microfluidization at different pressures resulted in modified casein-lipid structures that reflected the altered interactions of the smaller sized lipid droplets and intact or fractured casein micelles. Use of this technique will help meet consumer demand for novel dairy products such as cheeses with yogurt-like textures, cheese snacks, and desserts.