The objective of the present study was to test the hypothesis that the fracture strength of calcium hydroxide and mineral trioxide aggregate (MTA)-filled immature teeth decreased over time. Immature mandibular incisors from sheep were extracted and the pulps were extirpated using an apical approach with a barbed broach, and the teeth were divided into three experimental groups. Group 1: untreated teeth. Group 2: the root canals were filled with calcium hydroxide paste. Group 3: the root canals were filled with MTA. All specimens were kept in saline with 1% antibiotics at 4°C for certain periods of time: 2 weeks, 2 months, and 1 year. Then they were tested for fracture strength in an Instron testing machine. The results were subjected to statistical analysis by the Tukey–Kramer tests. A P-value (<0.05) was considered statistically significant. One tooth from each group was selected randomly for a histological study, examining matrix metalloproteinases (MMP2 and MMP14) and tissue inhibitor of metalloproteinase (TIMP). The results showed the mean fracture strengths decreased over time for all the three groups. Although the untreated teeth showed the highest value (45.5 MPa) at 2 weeks, the fracture strengths decreased significantly after 2 months (P < 0.05). On the other hand, the teeth treated with calcium hydroxide or MTA decreased, but not significantly over time (P > 0.05). For the MTA-treated teeth, the fracture strengths were not found significantly different from the untreated or calcium hydroxide-treated teeth at 2 weeks or 2 months (P > 0.05). However, the strength was significantly higher in the MTA group compared with the other two groups after 1 year (P < 0.05). Immunofluorescence images revealed expression of collagen type 1, MMP-2 and MMP-14 in both untreated and endodontically treated teeth. However, TIMP-2 was only observed in the MTA-treated teeth. In conclusion, the teeth with root treatment with MTA showed the highest fracture resistance at 1 year (P < 0.05). An explanation could be that MTA induced the expression of TIMP-2 in the dentin matrix and thereby possibly prevented destruction of the collagen matrix.