Advances in nanotechnology have empowered the design of bionanomaterials by assembling different types of natural biomolecules (e.g., nucleic acids, proteins, and lipids) as building blocks into nanoparticles (NPs) of 1-100 nm in diameter. Such bionanomaterials form the basis of useful nanomedicine applications, such as targeted delivery, gene regulation, molecular diagnostics, and immunomodulation. To achieve optimal performance in these applications, it is imperative that the NPs be delivered effectively to the organs, tissues, and cells of interest. A rational approach to facilitating the delivery of NPs is to develop a detailed and comprehensive understanding in their fundamental interactions with the biological system (or nano-bio interactions). Rigorous nano-bio research can provide mechanistic insights for circumventing the bottlenecks associated with inefficient and nonspecific delivery of NPs, catalyzing the clinical translation of nanomedicines. Cationic liposomes and lipid NPs are conventional carriers of therapeutic cargoes into cells due to their high ability to penetrate the cell membrane, a barrier comprised by an anionic phospholipid bilayer. Yet, cationic NPs tend to cause cytotoxicity and immune responses that may hamper their clinical translation. Contrary to cationic NPs, non-cationic NPs (be they near-neutral or anionic in surface charge) generally exhibit higher biocompatibility but enter mammalian cells in much less pronounced amounts. Intriguingly, some types of non-cationic NPs exhibit high biocompatibility and cellular uptake properties, all attractive features for intracellular delivery. In this Account, we present our studies of the interactions of non-cationic bionanomaterials with cells (or nano-cell interactions). To start with, we introduce the use of near-neutral poly(ethylene glycol)-coated NPs for probing the roles of two rarely explored physicochemical parameters on cellular uptake, namely, extracellular compression and alkylation. We next present the nano-cell interactions of two representative types of anionic bionanomaterials that effectively enter mammalian cells and have found widespread applications in the past decade, including DNA-coated NPs and polydopamine (PDA)-coated NPs. In our cell-based studies, we dissect the route of intracellular trafficking, pathway proteins that dictate cellular uptake, and trafficking of NPs. We further touch on our recent quantitative analysis of the cellular-level distribution of NPs in various organs and tissues of diseased animal models. Our results offer important design rules of NPs for achieving effective intracellular delivery and may even guide us to explore nanomedicine applications that we did not conceive before, such as using DNA-coated NPs for targeting atherosclerotic plaques and PDA-coated plasmonic nanoworms for photothermal killing of cancer cells. We conclude with our perspectives in elucidating nano-bio interactions via a reductionist approach, calling for closer attention to the role of functional groups and more refined studies on the organelle-level distribution of NPs and the genetic basis of in vivo distribution of NPs.