The transport of fluid in and around nanometer-sized objects with at least one characteristic dimension below 100 nm enables the occurrence of phenomena that are impossible at bigger length scales. This research field was only recently termed nanofluidics, but it has deep roots in science and technology. Nanofluidics has experienced considerable growth in recent years, as is confirmed by significant scientific and practical achievements. This review focuses on the physical properties and operational mechanisms of the most common structures, such as nanometer-sized openings and nanowires in solution on a chip. Since the surface-to-volume ratio increases with miniaturization, this ratio is high in nanochannels, resulting in surface-charge-governed transport, which allows ion separation and is described by a comprehensive electrokinetic theory. The charge selectivity is most pronounced if the Debye screening length is comparable to the smallest dimension of the nanochannel cross section, leading to a predominantly counterion containing nanometer-sized aperture. These unique properties contribute to the charge-based partitioning of biomolecules at the microchannel-nanochannel interface. Additionally, at this free-energy barrier, size-based partitioning can be achieved when biomolecules and nanoconstrictions have similar dimensions. Furthermore, nanopores and nanowires are rooted in interesting physical concepts, and since these structures demonstrate sensitive, label-free, and real-time electrical detection of biomolecules, the technologies hold great promise for the life sciences. The purpose of this review is to describe physical mechanisms on the nanometer scale where new phenomena occur, in order to exploit these unique properties and realize integrated sample preparation and analysis systems.