With the recent development of 3D printing technology, concrete materials are sometimes used in 3D printing. Concrete structures based on 3D printing have been characterized to have the form of multiple layer build-up. Unlike general concrete structures, therefore, the 3D-printed concrete can be regarded as an orthotropic material. The material property of the 3D-printed concrete's interface between layers is expected to be far different from that of general concrete bodies since there are no aggregate interlocks and weak chemical bonding. Such a difference finally affects the structural performance of concrete structures even though the interfaces are formed before initial setting of the concrete. Furthermore, it was confirmed that superimposed load from the accumulated layers increase the fracture energy of interface between two layers. The current study mainly reviewed the changes in fracture energy (toughness) with respect to various environmental conditions of such interface. Changes in fracture energies of interfaces between concrete layers were measured using low-speed Crack Mouth Opening Displacement (CMOD) closed loop concrete fracture test. The experimental results indicated reduction in fracture energy as well as tensile strengths. In order to improve the tensile strength of interfaces, use of bridging materials are suggested. Since it was assumed that reduction in fracture energy could be a cause of shear strength, in order to evaluate the reduced structural performance of concrete structure constructed with multiple interfaces by 3D printing technology, shear strength of RC beam by 3D printing technology was predicted and compared with plain RC beam. Based on the fracture energy measured in this study, MCFT theory-applied Vector 2 program was employed to predict the degree of reduction in shear strength without considering stirrups. Reduction factors were presented based on the obtained results to predict the reduction in shear strength due to interfaces before initial setting of the concrete.