Many studies on brittle fracture initiation mechanisms have focused on Ferrite-Pearlite steel. Recently, high-strength steel has attracted attention in order to achieve cost saving. Since the 1970’s, Bainite microstructure has been commonly used for structural high-strength steel instead of Ferrite-Pearlite steel. In engineering, it is important to understand the mechanism of brittle crack initiation of Bainite as well as that of conventional Ferrite-Pearlite. However, due in part to the complexity of the Bainite microstructure, its micro-mechanism has not been revealed. In this study, six laboratory-scale steels with a composition of 0.2%C-2%Mn-1.5%Ni were prepared in order to clarify the qualitative and quantitative effects of several candidate controlling factors on brittle crack initiation behavior, which are phase transformation, prior austenite grain size, Martensite-Austenite constituent amount and size, cementite shape and size and orientation information inside the prior austenite grain. Charpy impact tests were used for analysis of the brittle crack initiation conditions. The results suggest that the most effective key factor in degrading the brittle crack initiation behavior is the existence of the tempered Martensite-Austenite constituent on the prior austenite grain boundary.