Treatment and management of tracheal defects remain challenges in head and neck surgery. The purposes of this study were to explore a novel strategy to fabricate tissue-engineered trachea by using chondrocyte macroaggregate, and evaluate the feasibility of creating tracheal cartilage equivalents grown in the shape of cylindrical structure without scaffold. Chondrocytes from rabbit cartilage were expanded and seeded into a culture dish at high density to form mechanically stable chondrocyte macroaggregate. Once the chondrocyte macroaggregate was harvested by scrapping technique, it was wrapped around a silicon tube and implanted subcutaneously into the cell donor rabbit. Eight weeks later, specimens were harvested and analyzed for gross appearance, and histological, biochemical, and biomechanical properties. These values were compared with native rabbit cartilage. It was found that expanded chondrocytes could be harvested as a coherent cellular macroaggregate and could be fabricated into a tubelike graft. After in vivo implantation, cartilage-like tissue with cylindrical structure was regenerated successfully. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans; type II collagen was also highly expressed in this engineered trachea cartilage. In a conclusion, based on the chondrocyte macroaggregate strategy, tracheal cartilage equivalents with cylindrical shape could be successfully reconstructed. This construct has advantages of high cell-seeding efficiency, good nutritional perfusion, and minimal inflammatory reaction, which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea-cartilage loss encountered in clinical practice.