When introduced into biological systems, the function and biodistribution of lipid nanoparticles (LNPs) are affected by the biomolecular coronas they acquire. Corona composition is determined by the biophysical and chemical properties of the particles and the contents of the biofluids. Polyethylene glycol (PEG) polymers, anchored using lipids that partition into LNPs during formulation, are key to LNP stability in circulation. It is, however, not well-studied how different PEG-lipid anchors, with different acyl chain lengths, headgroup/linker chemistries, and desorption rates (PEG “shedding” from nanoparticles) can affect corona composition and LNP function. Here, we examined how common PEG-lipid anchors affect (1) in vivo biodistribution in C57BL/6 mice, (2) corona content (using mass spectrometry-based proteomics), (3) LNP biophysical characteristics (using single-particle automated Raman trapping analysis (SPARTAⓇ)), and (4) in vitro particle function (using cellular uptake and cargo delivery assays). Following nanoparticle formulation with clinically approved, commonly used PEG anchors, we found that the LNP biodistribution is strongly impacted, particularly in the liver, spleen, bone marrow, and lung. We then tested a wide range of lipid ratio combinations using high-throughput evaluation in vitro. Despite being minor LNP components (by molar ratio), the PEG-lipid anchors strongly impact the chemical characteristics, corona content, and particle function. These findings reveal structure–activity relationships between PEG-lipid anchor chemistry and functional LNP biodistribution, with implications for rational LNP design.