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.