The flow behavior of concrete is highly impacted by the inherent structure of the paste matrix, which in turn is governed by the aggregation mechanisms within the paste matrix. Further, the mixing process is an essential process of cement-based materials preparation that influences the rheology of cement paste via the microstructure formation of cement paste. Due to the difficulty of measuring the rheology of concrete with aggregates, cement paste is used to represent the rheology of concrete. Based on literature in this area of research [1], it is known that a faster mixing speed is appropriate to simulate the condition of the cement paste inside of the concrete mixture during mixing. In 2011, the American Society for Testing and Materials (ASTM) published a new standard for high-shear mixing of hydraulic cement paste (ASTM C1738, “Standard Practice for High-shear Mixing of Hydraulic Cement Pastes,” 2011) to provide guidance for preparation of cement paste samples for rheological studies in hydraulic cement systems. Despite the improvements gained in the implementation of several hydraulic cement paste standards for mixing throughout the years, the relationship between the rheology and fresh state microstructure of cement paste with different mixing intensities—especially those with a very high mixing intensity range is not known yet. Overall, there is a lack of fundamental knowledge about the influence of the applied mixing forces on the internal structure of cement paste, the role of the microstructure on the rheological behavior, and microstructure formation mechanisms on rheological behavior. The objectives of this research are to (1) evaluate the influences of sample preparation on the rheology of cement paste, (2) analyze the influence of the mixing intensity on rheological behaviors, (3) characterize the microstructure of fresh state cement paste (4) understand the mechanisms of determining cement agglomerate size and identify the relationship between the microstructure and rheology of fresh state cement paste. In order to accomplish these objectives, rheology studies on cement pastes mixed with different mixing intensities were conducted. Based on the rheology studies with a high mixing intensity, it was found that increasing the mixing intensity does not always result in a reduction in the rheological properties. Rather interesting results can occur when a high-range water reducer is incorporated, and possible explanations for this unexpected behavior are presented. To prove the reasons for this unexpected result, two hypotheses were proposed: (1) If cement paste has increased ionic concentration under the high mixing intensity conditions, then the cement flocs are aggregated (flocculated), and thus these aggregated cement flocs likely contribute to the increased apparent viscosity. (2) If the polycrboxylate-based high-range water reducer (HRWR) produces unexpected air bubbles under the high mixing intensity conditions, then those air bubbles will have an influence on increasing apparent viscosity. To prove these hypotheses, a series of experiments were conducted, and based on the results of these experiments microstructural formation mechanisms were suggested to explain the unexpected flocculation under the high mixing intensity, and the numerical relationship was investigated between the microstructure and rheology properties of fresh state cement paste. In this research, the relationship between microstructural change and rheological behaviors of fresh state cement paste were investigated and a better understanding of the mechanisms of microstructural formation with various mixing intensity conditions for cement-based materials was obtained.