In this study, cobble-stone (CS) and broke-stone materials were used as filter media in horizontal sub-surface flow constructed wetlands (HSSF-CWs) for highway runoff treatment. These wetlands were planted with Phragmites spp. plants. The Lukou viaduct section of the Nanjing airport expressway was selected for collection of highway runoff. The HSSF-CWs were subjected to two hydraulic retention times (HRT) of 12 and 24 h with different hydraulic loads and influent flow rate. Based on the results, the CW with broken-stone (BS) substrate achieved significantly higher removal efficiency for SS (75 and 55%), chemical oxygen demand (45 and 50%), NH3-N (78 and 88%), and total phosphorus (77 and 34%) in 12 and 24 h HRT compared with CS substrate wetlands. The CW with CS substrate showed lower removal efficiencies than the BS substrate wetland. The removal efficiencies for Cu, Zn, and Pb for the 12 h HRT were 62, 80, and 9%, respectively, and the concentrations of Cu, Zn, Pb, and Cr were generally under the standard limits. This work will aid design and improvement of constructed wetlands for small scale treatment of highway runoff.