ABSTRACT Objective: This in vitro study analyzed the effects of a bleaching treatment containing 35% hydrogen peroxide (HP) with or without calcium on bovine enamel, using the Knoop hardness number (KHN), tristimulus colorimetry (TC), and scanning electron microscopy. Materials and Methods: Forty-five specimens were randomly divided into groups (n = 5), which included artificial saliva (negative control [NC]), 35% HP (positive control [PC]), and 35% HP Blue Calcium (HP Blue). The specimens were subjected to three bleaching sessions. During the sessions, the specimens were immersed in artificial saliva at 37°C. Before and after bleaching, KHN tests were conducted using a force of 25 gf for 5 s. TC was performed using the CIE-L*a*b* system and readouts were obtained at the following 4 time points: Before the bleaching treatment; after the first session, the second session, and the third session. The specimens were dehydrated and coated with gold, and the photomicrographs were analyzed in a double-blind manner with a LEO microscope. Results: Using one-way analysis of variance and Tukey's test (P < 0.05), a statistically significant difference was identified between the initial and final mean KHNs of the NC and PC groups, while the initial and final mean KHNs were not significantly different in the HP Blue group. The final mean values of ⋄E, ⋄L, and ⋄b of the PC and HP Blue groups were significantly higher than the initial values (P < 0.01 for both). The photomicrographs revealed no differences among the groups. Conclusions: Therefore, treatment with HP Blue prevented changes in the KHN without reducing the efficacy of bleaching.