Fatigue cracking in steel girder web penetration details is so dangerous that it can break steel girders. A one-meter-long crack was detected in Yamazoe Bridge in 2006. Since a number of highway bridges with such web penetration details may exist in Japan, it is of urgent importance to understand these fatigue-strength properties. However, few fatigue tests have been reported on steel girder web penetration details. The purpose of this study is to clarify fatigue behavior of steel girder web penetration details with a slit through fatigue tests of specimens with these details. We designed and fabricated girder specimens that have steel girder web penetration details, in which cross-beam bottom flanges are connected to each top or bottom surface of a slit by welding. First, we conducted static loading tests to understand the stress distributions around web penetration details. Second, we conducted fatigue tests to examine fatigue crack initiation and propagation behavior and fatigue strength.