The aim of this study is to investigate the impact of global operating parameters, e.g., engine speed, brake mean effective pressure, and air–fuel ratio, of a turbocharged 4-cylinder GDI engine on the reactivity of soot particles against oxidation. The knowledge of soot reactivity is crucial for optimizing gasoline particulate filter regeneration strategies and is, consequently, a key parameter for reducing fuel consumption and CO2 emissions. In this work, time-resolved in-cylinder soot concentrations and exhaust particle size distributions are measured by using two-color pyrometry, engine exhaust particle sizer and smoke meter, respectively. Reactivity against oxidation by molecular oxygen is determined by temperature programmed oxidation analysis. To derive a physicochemical explanation for varying soot reactivity, the morphological and nanostructural primary particle structure of collected samples is analyzed using high-resolution electron microscopy and image analysis algorithms. The results reveal that engine operating parameters affect soot reactivity differently. While engine speed has only a slight effect, a reduction of air/fuel ratio (λ < 1.0) or an increase of BMEP > 10 bar significantly reduces the soot oxidation reactivity. These findings give evidence, that the quality of the fuel/air mixture is a significant parameter influencing soot reactivity. Measured soot concentrations substantiate the hypothesis that low-sooty homogeneous premixed combustion of a homogeneous fuel/air mixture favors formation of high-reactive soot particle fractions. Reactive soot particle aggregates are composed of multiple soot fractions of different reactivity. Reactive primary particles are composed of short graphene-like layers and vice versa, providing a physicochemical explanation for varying soot reactivity depending on engine operating conditions.