In this study, a series of small-scale experiments was carried out in a model scale tunnel with dimensions of 20 m (Length) × 2 m (Width) × 1 m (Height) to investigate the characteristics of buoyancy-driven smoke flow in a tunnel with vertical shafts. Different shaft settings and four different longitudinal ventilation velocities were tested in the experiments. A theoretical model for the mass flow rate of buoyancy-driven smoke flow in the shaft was developed and validated. The gas temperature along the tunnel ceiling and smoke stratification were subsequently analyzed and discussed. The results showed that more shafts, greater shaft heights and greater shaft cross sectional areas can significantly increase the smoke extraction rate, and the total smoke mass flow rate in the shafts increases with the increasing ventilation velocity. The local pressure loss coefficient at the shaft inlet may not be a fixed value. An average value of 1.0 for this coefficient was recommended for engineering estimation and design of rectangular-shaped natural shafts. The presence of vertical shaft is beneficial to the smoke stratification and could increase the height of the smoke layer interface, especially for the downstream of the shaft.