Objective The aim of this research was to check the effect of the prenatally administered β-hydroxy β-methylbutyrate (HMB) on the development of enamel surface of the spiny mice offspring. Design The spiny mice dams were randomly assigned into three groups: control group (not supplemented with HMB) and two experimental groups in which powdered HMB was given at the daily dosage of 0.2 g/kg of body weight (group I) and 0.02 g/kg of body weight (group II) during the last period of gestation. Newborn pups were euthanized by CO2 inhalation. The morphology of incisor teeth was analysed using atomic force microscopy (AFM) in semi-contact mode in the height, magnitude and phase domains. Height images became a basis for determination of surface roughness parameters. Results Conducted study indicated that maternal HMB administration markedly influences enamel development. Enamel of offspring’s teeth in both experimental groups was characterized by significantly smaller values of indices describing surface roughness and profile. HMB supplementation influenced the calculated parameters regardless of the diet type and offspring sex, however higher dose of HMB caused stronger changes in enamel surface’s physical properties and could be observed in higher intensity in the male group. Conclusions HMB administration caused reduction in the irregularities of enamel surface, thereby possibly reducing the probability of bacteria adhesion and caries development. These observations may serve to improve nutrition and supplementation of animals and could be a lead for further research.