<p>Manufacturing-induced pre-strain has a significant influence on high-cycle fatigue (HCF) behavior in structural steels. Fatigue tests at different stress amplitudes were conducted after tensile pre-strain levels of 0%, 2.5%, 5.0%, and 7.5%. The present results reveal a pronounced stress-amplitude-dependent effect of pre-strain. At high stress amplitudes, pre-strain accelerates fatigue failure and reduces fatigue life, whereas at low stress amplitudes, pre-strain enhances fatigue resistance and extends fatigue life. This contrary response is attributed to a competition between damage activation and strengthening retention, where pre-strain-induced heterogeneity promotes localized microplasticity and early damage accumulation under high stress amplitudes, while the partial rearrangement and stabilization of the dislocation structure and its inhibitory effect on intense local slip bands become dominant under low stress amplitudes. Under different stress amplitudes and pre-strain levels, EBSD-based orientation analysis, surface morphology characterization, and fractographic observations were employed to characterize pre-strain-induced deformation and strain heterogeneity, identify potential crack-initiation sites, and reveal the stress-amplitude-dependent characteristics of crack initiation and propagation. Based on these findings, a pre-strain-modified equivalent stress amplitude model is proposed to improve the accuracy of fatigue life prediction. This study provides valuable support for understanding and modeling the stress-amplitude-dependent effects on fatigue life of pre-strained structural steels.</p>