The emergence of mRNA-containing lipid nanoparticles (LNPs) during the COVID-19 pandemic has revolutionized vaccine technology and is now being explored for protein replacement therapies. This study aimed to test the hypothesis that LNP size, independent of lipid composition, critically influences their physicochemical and biological performance. To achieve this, we employed computational fluid dynamics (CFD) simulations to predict the mixing index within a microfluidic channel, identifying the flow rate conditions at which a 70% mixing index was reached. By tuning the flow rate accordingly, we generated LNPs across a controlled size range of 30-270 nm while maintaining identical lipid ratios. We systematically characterized these LNPs for zeta potential, pKa, generalized polarization, and hemolytic activity. Cellular studies in HeLa cells revealed that smaller LNPs exhibited higher uptake and transfection efficiency, which was quantitatively explained using a mathematical model of cellular uptake. In vivo studies further demonstrated that LNP size affected both the magnitude of mRNA expression and the biodistribution across organs following intravenous and intramuscular administration. Our findings demonstrate precise flow rate-induced size control of LNPs via microfluidics and reveal that smaller LNPs achieve superior gene expression in vitro and exhibit preferential transfection in vivo, underscoring the importance of size as a design parameter for mRNA delivery systems.