Achieving extensive laminar flow on engine nacelle surfaces to reduce drag has emerged as a crucial strategy for improving the economic performance of large aircraft. This paper investigates a self-optimized natural laminar flow nacelle through combined cryogenic wind tunnel testing and numerical simulations, systematically exploring the Reynolds number effects on transition characteristics and underlying mechanisms. The results demonstrate that wall temperature variations within 5–10 °C induced by heating power show negligible influence on transition location. The cryogenic Temperature-Sensitive Paint (TSP) technique incorporating electrically heated coatings proves effective in achieving reliable transition visualization while significantly enhancing testing efficiency. As the Reynolds number increases, surface pressure distribution remains relatively stable, while both momentum thickness Reynolds number (Reθt) and surface friction coefficient experience accelerated growth. This leads to earlier attainment of critical momentum thickness Reynolds numbers (Reθc) in the boundary layer near the nacelle lip, consequently advancing transition onset. Quantitative analysis reveals a transition location movement rate of approximately 2% per million Reynolds number increment within medium-low Reynolds number regimes (Re ≤ 5 × 106). At elevated Reynolds numbers, thinning boundary layers exhibit heightened sensitivity to wind tunnel contaminants and surface roughness, manifested through expanded wedge-shaped turbulent regions. Furthermore, microscopic imperfections in pressure tap installations are found to induce localized premature transitions. These phenomena collectively underscore the imperative for stringent surface quality control in cryogenic high-Reynolds-number laminar flow experiments. The study provides critical insights into Reynolds number effects on nacelle boundary layer stability, offering valuable guidance for aerodynamic design optimization of next-generation laminar nacelles.