Mucus is an endogenous viscoelastic biopolymer barrier that limits the entry of foreign pathogens and therapeutic carriers to the underlying mucosal cells. This could be overcome with a hydrophilic and nonpositively charged carrier surface that minimizes interactions with the mucin glycoprotein fibers. Although PEGylation remains an attractive surface strategy to enhance mucopenetration, cell uptake of PEGylated nanoparticles (NPs) often remains poor. Here, we demonstrated polydopamine (PDA) coating to enhance both mucopenetration and cell uptake of NPs. PDA was polymerized on carboxylated polystyrene (PS) NPs to form a PDA coating, and the resulting PS-PDA achieved a similar level of mucopenetration as our PEGylated PS (PS-PEG) positive control in three separate studies: NP–mucin interaction test, transwell assay, and multiple particle tracking. Compared to water, the diffusions of PS-PDA and PS-PEG in reconstituted mucus solution were only 3.5 and 2.4 times slower, respectively, whereas the diffusion of bare PS was slowed by up to 250 times. However, the uptake of PS-PDA (61.2 ± 6.1%) was almost three times higher than PS-PEG (24.6 ± 5.4%) in T24 cells, which were used as a model for underlying mucosal cells. Our results showed a novel unreported functionality of PDA coating in enhancing both mucopenetration and cell uptake of NPs for mucosal drug delivery applications, not possible with conventional PEGylation strategies.