155 kinds of concrete with different grading of fine aggregate and different mix proportion such as water-cement ratio, unit water content and sand-total aggregate ratio were mixed by the subcommittee for revision of Recommendation for Practice of Mix Design of Concrete organized in Architectural Institute of Japan. Additionally, a slump test and a cylinder penetration test for them and a flow test (JIS R 5201) for their wet-screened mortar were conducted. Furthermore, the following considerations were conducted by applying the excess paste theory, which can comprehensively evaluate the effects of grading of aggregate, aggregate content, sand-total aggregate ratio, etc. on fluidity of concrete, to the experimental results: (1) the effect of grading of fine aggregate on workability of concrete, (2) the optimum combination of unit water content and sand-total aggregate ratio based on the index to evaluate segregation of concrete, (3) the procedure of mix design more reasonable than ever before. The following findings were obtained as the results. (1) A slump value is determined by the combination of unit water content and sand-total aggregate ratio in principal. Besides, the area that a slump value hardly changes with sand-total aggregate ratio in case of constant unit water content exists in the relationship between them, and the area is wider as unit water content increases. (2) The segregation degree estimated by observing the state of fresh concrete of the slump test can be evaluated quantitatively by an inflow mortar value, the result of the cylinder penetration test. Moreover, the inflow mortar value corresponding to the limit of segregation of concrete is 30 mm. (3) The fluidity of matrix mortar has a dominant influence on the segregation degree of concrete. Moreover, the threshold of the flow value, that is the measure without falling motion, of wet-screened mortar corresponding to the limit of segregation of concrete is 160 (≈163). (4) Depending on grading of fine aggregate and mix proportion, the concrete with a given slump value and the minimum unit water content may segregate, and in that case, the optimum combination of unit water content and sand-total aggregate ratio need to be determined with the aim of ensuring segregation resistance of concrete. (5) In light of the considered results in this study, the following procedure of mix design of concrete is reasonable: First, sand-cement ratio and dosage of chemical admixture of matrix mortar should be set so that its flow value is that (160 + the difference of flow value between wet-screened mortar and ordinary mortar) corresponding to the limit of segregation of concrete. Next, the ratio of coarse aggregate to matrix mortar should be set so that a given slump value of concrete is obtained.