This study aimed to determine the effect of iontophoresis delivery of fluoride in stannous fluoride (SnF2) toothpaste on dentin permeability in human extracted third molars. For dentin permeability test, 26 dentin specimens were randomly divided into 4 groups; SnF2 without-iontophoresis (n = 10), SnF2 with-iontophoresis (n = 10), no SnF2 without-iontophoresis (n = 3), and no SnF2 with-iontophoresis (n = 3). The hydraulic conductance of dentin (HD) was measured after smear layer removal, immediate treatment, 7 days, and acid challenge. The other 26 specimens were also prepared under these different conditions to assess degree of dentinal tubule occlusions using scanning electron microscopy (SEM). Percentage decrease of HD in SnF2 without-iontophoresis after immediate treatment, 7 days and acid challenge were 38.38 ± 13.61, 56.92 ± 17.22 and 33.07 ± 23.57%. The corresponding values in SnF2 with-iontophoresis were 42.16 ± 14.49, 62.35 ± 15.67 and 50.01 ± 12.60%, respectively. There was a significant difference between without- and with-iontophoresis groups after acid challenge (p < 0.05). For SEM, after 7 days, SnF2 with-iontophoresis showed deeper dentinal tubule occlusion (p < 0.05) and more resistance to acid challenge than SnF2 without-iontophoresis. No significant change was observed in groups of no SnF2 treatment. Cathode iontophoresis could enhance the effect of SnF2 toothpaste in decreasing dentin permeability and increasing resistance to acid challenge.