Background: This study was designed to investigate the effects of the vitamins C and B12 on the regulation of human nasal ciliary beat frequency (CBF). Methods: Human nasal mucosa was removed endoscopically and nasal ciliated cell culture was established. Changes of CBF in response to different concentrations of vitamin C or vitamin B12 were quantified by using high-speed (240 frames per second) digital microscopy combined with a beat-by-beat CBF analysis. Results: At the concentrations of 0.01% and 0.10%, vitamin C induced an initial increase, followed by a gradual decrease of CBF to the baseline level, while 1.00% vitamin C induced a reversible decrease of CBF. Vitamin B12, at the concentrations of 0.01% and 0.10%, did not influence CBF during the 20-min observation period, while a 1.00% vitamin B12 treatment caused a time-dependent but reversible decrease of CBF. Conclusions: Treatment with vitamin C or vitamin B12 caused a concentration-dependent but reversible decrease of CBF in cultured human nasal epithelial cells. Therefore, it is necessary to choose a concentration that is safe, effective, and non-ciliotoxic when applying these drugs topically in the nasal cavity.