Shallow trophoblast invasion is detrimental in human pregnancies, but represents normal endotheliochorial placentation in dogs. Factors regulating shallow trophoblast invasion into the canine decidua are not well described, but it is known that matrix metalloproteinases (MMPs) play a crucial role in trophoblast invasion in many species. Following the methods previously described for isolating human trophoblasts, canine trophoblasts were isolated using collagenase and trypsin digestions with Percoll density gradient centrifugation. In addition, placental pieces were cryopreserved prior to primary culture following methods previously described for human tissue. Expression of cytokeratin-7, MMP2 and MMP9 was confirmed using fluorescent immunocytochemistry. Cellular morphology was similar to that reported for trophoblasts. More than 97% of the cells cultured expressed cytokeratin-7. More cultured canine trophoblasts expressed MMP9 (54.7 ± 3.4%) compared with MMP2 (40.3 ± 1.8%) (p = 0.02). Although both MMPs were immunolocalized to the cytoplasm, MMP2 was found in large, coalescing granules, whereas MMP9 was more diffusely expressed throughout the cell. Cryogenic freezing of placental tissue prior to primary cell culture had no effect on cell proliferation (p = 0.37). This research has established a baseline for future studies investigating the canine placenta as a model for disorders of shallow trophoblast invasion in humans.