Nanoparticle-based therapeutics and diagnostics are commonly referred to as nanomedicine and may significantly impact the future of healthcare. However, the clinical translation of these technologies is challenging. One of these challenges is the efficient delivery of nanoparticles to specific cell populations and subcellular targets in the body to elicit desired biological and therapeutic responses. It is critical for researchers to understand the fundamental concepts of how nanoparticles interact with biological systems to predict and control in vivo nanoparticle transport for improved clinical benefit. In this overview article, we review and discuss cellular internalization pathways, summarize the field`s understanding of how nanoparticle physicochemical properties affect cellular interactions, and explore and discuss intracellular nanoparticle trafficking and kinetics. Our overview may provide a valuable resource for researchers and may inspire new studies to expand our current understanding of nanotechnology-biology interactions at cellular and subcellular levels with the goal to improve clinical translation of nanomedicines.