Abstract: Objectives To explore the experimental approach for in vitro primary culture of chondrocytes of mouse cranial base synchondrosis and to investigate its basic biological features. Methods The spheno-occipital synchondrosis of new natal mouse was harvested through surgical dissection, from which chondrocytes were further isolated through enzymatic digestion with trypsin and type II collagenase. Chondrocytes of the primary and successive generations were cultured with serum containing Dulbeccomodified eagle's medium (DMEM) . Optical microscopic imaging, growth curve tracing and immunohistochemistry were conducted to observe the morphological features, growth behavior and type II collagen expression. Results Within five generations, the cultured cells consistently presented typical morphological and functional features of chondrocyte. Conclusion The surgical tissue dissection combined with double enzymatic digestion is a reliable experimental approach for the primary culture of chondrocytes of mouse cranial base snychondrosis.