Background <p>Toxoplasmosis is a globally distributed zoonosis, caused mainly by the ingestion of contaminated water and food. <i>Toxoplasma gondii</i> is the protozoan that causes the disease and can infect humans by forming cysts in the tissues of its hosts. The parasite can also cross the transplacental barrier and infect the fetus, with consequences for embryonic development. Patients infected with <i>T. gondii</i> have muscle inflammation and polymyositis. Most of the knowledge regarding <i>T. gondii</i> infection in skeletal muscle tissue has been established from few <i>in vivo</i> models and 2D cell culture. Three-dimensional cultures have increasingly been used as preclinical models to study the pathogenesis of several diseases, including cancer, muscle dystrophies and infectious diseases. Herein we describe a new three-dimensional culture model using embryonic chick skeletal muscle cells to study the effects of <i>T. gondii</i> infection on the development of skeletal muscle tissue.</p> Methods <p>Pectoralis muscle from 11-day-old chicken embryos was used as a source for primary cultures and subsequent 3D spheroids formation. The dynamic formation of chick spheroids was followed by real-time video microscopy and its morphology by confocal fluorescence microscopy and scanning electron microscopy. The levels of myosin heavy chain (MyHC) and viculin were also assessed by western blotting.</p> Results <p>Spheroids made of embryonic chick skeletal muscle cells have an ultrastructural organization and morphology similar to that observed in muscle tissues <i>in vivo</i>. <i>T. gondii</i>-infected spheroids showed a decrease in their area and formation of muscle fibers compared to non-infected control spheroids, with reduced MyHC and vinculin protein levels. These results highlight the use of chick skeletal muscle cell spheroids as a new promising experimental model for studies of <i>T. gondii</i> infection in muscle cells.</p>