Type VI secretion system (T6SS) is a contact-dependent secretion system, employed by most gram-negative bacteria for translocating effector proteins to target cells. The present study was conducted to investigate T6SS in Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice, and to unveil its functions. Two T6SS clusters were found in the genome of Xoo PXO99A. The deletion mutants, Δhcp1, Δhcp2, and Δhcp12, targeting the hcp gene in each cluster, and a double-deletion mutant targeting both genes were constructed and tested for growth rate, pathogenicity to rice, and inter-bacterial competition ability. The results indicated that hcp in T6SS-2, but not T6SS-1, was involved in bacterial virulence to rice plants. However, neither T6SS-1 nor T6SS-2 had any effect on the ability to compete with Escherichia coli or other bacterial cells. In conclusion, T6SS gene clusters in Xoo have been characterized, and its role in virulence to rice was confirmed.