The use of nanoparticles (NPs) can improve drug delivery in many pharmaceutical applications. Compared to conventional compounds, NPs are expected to show better tissue penetration and to deliver higher drug amounts more selectively to the target site. NP-based formulations are most advanced for parenteral applications but they are also used for delivery across mucus-covered epithelial surfaces (eye, orogastrointestinal tract, airways, and vagina). The mucus layer represents an important barrier for an NP-based delivery system. In this review, mucus composition, architecture, and turnover of the mucus layer at different anatomical locations are addressed. The influence of particle parameters on mucus penetration/permeation is mentioned and examples for mucoadhesive, mucolytic, and mucus-penetrating particle systems are listed. While mucoadhesive particles have a relatively long history in NP-based drug delivery, mucus penetrating NPs have been developed more recently. These particles may be advantageous for drug delivery to anatomical sites with high mucus turnover.