Purpose: To evaluate the effects of desensitizing MI Varnish (GC America) applied before or after bleaching on the mineral component of enamel and surface topography. Materials and Methods: The coronal portions of 10 freshly extracted bovine teeth were segmented for a total of 40 specimens. Enamel specimens obtained from each tooth were randomly divided into four groups (n = 10): Group A = no bleaching; Group B = bleaching with 40% hydrogen peroxide (HP); Group C = MI Varnish applied before bleaching; and Group D = MI Varnish applied after bleaching. The calcium (Ca) and phosphorus (P) levels of the specimens in each group were determined by energy dispersive spectroscopy (EDS). Morphologic changes were observed using scanning electron microscopy (SEM). One-way ANOVA and Tukey HSD tests were used for statistical analyses (α = .05). Results: The mean Ca content of Group B was significantly lower than those of Groups A, C, and D (P < .05). The mean Ca content of Group C was significantly lower than that of Group A (P < .05). There was no significant difference in Ca content between the other groups (P > .05). The mean P content of Group A was significantly higher than those of Groups B to D (P < .05). There was no significant difference in P content between Groups B to D (P > .05). Conclusions: Application of MI Varnish before or after in-office bleaching was effective in reducing mineral loss. However, applying MI Varnish after bleaching was more effective.