The low cycle fatigue tests using the replica technique for LZ50 steel under non-proportional cyclic loading were carried out, and eight groups of effective test data were obtained. The evolution behaviour of short cracks was studied based on the effective short cracks criterion. The results show that short cracks generally originate in the grain or along the grain boundary. At the microstructural short crack stage, the crack propagation is influenced strongly by the microstructure of the material, and the growth rate of the short crack slows down several times according to the number of obstacles encountered. At the physical short crack stage, the crack propagation breaks through the banded structure of pearlite. Thus, the dominant effective short fatigue crack is formed, and the crack growth rate increases rapidly. Based on the modified parameters of the uniaxial short crack model, an approach is presented to calculate the growth rate of short cracks under multi-axial non-proportional loadings, and the new model can consider the non-proportional factor F. The fitting results of the multi-axial microstructural obstacles model are compared with test data. The comparison results show that this model can reflect the trend of short fatigue crack propagation rate under non-proportional loadings.