Objective: Accurate sex estimation is an essential step for the reconstruction of the biological profile of human remains. Earlier studies have shown that elements of the human permanent dentition are sexually dimorphic. The aims of this study are to determine the degree of sexual dimorphism in the dental tissue volumes and surface areas of mandibular canines and to explore its potential for reliable sex determination. Method The teeth included in this study (n = 69) were selected from anthropological collections from Spain, South Africa and Sudan. In all cases, the sex of the individuals was known. The teeth were scanned and three-dimensional (3D) measurements (volumes and surfaces areas) were obtained. Finally, a dsicriminant function analysis was applied. Results Our results showed that sexual dimorphism in canine size is due to males having greater amounts of dentine, whereas enamel volume does not contribute significantly to overall tooth size dimorphism. Classification accuracy of the multivariable equations tested on slightly worn teeth ranged from 78 to 90.2% for the crossvalidation, and from 71.43 to 84.62% for the hold-out sample validation. When all functions were applied together, the sex was correctly assigned 92.30% of the time. Conclusions Our results suggest that the 3D variables from mandibular canine dental tissues are useful for sex determination as they present a high degree of dimorphism. The results obtained show the importance of 3D dental tissue measurements as a methodology in sex determination, which application should be considered as a supplemental method to others.