Steel structures are frequently exposed to the environment with variable temperature, where the fatigue failure of steel is difficult to be predicted. In this paper, the standard Q420C high-strength steel specimens were tested to explore their fatigue performance at room (25°C) and low temperatures (0°C, −15°C and −30°C). Before fatigue tests, the tensile tests have been done, and the results indicate that the yield and ultimate strengths of Q420C steel significantly grew when the temperature decreased. The fatigue behavior of Q420C steel was discussed from different perspectives, including the crack initiation, crack growth, fracture mode and fatigue life. The fatigue failure was observed mainly near the arc transition section of specimen, with the fatigue crack initiated from the surface and internal discontinuity defects. Furthermore, the test fatigue life was compared with the recommended S-N curves provided by specifications from various countries. These comparison results suggest that recommended S-N curves were excessively conservative to evaluate the steel fatigue life at low temperature, leading to a potential economic cost for the steel structures at extremely cold conditions. To avoid needless loss, a calculation method of steel fatigue life considering the effect of low-temperature, stress range and yield strength was proposed.