Iron is one of the most important elements for bacterial survival and pathogenicity. The iron uptake mechanism of Riemerella anatipestifer (R. anatipestifer, RA), a major pathogen that causes septicemia and polyserositis in ducks, is largely unknown. Here, the functions of the putative TonB-dependent iron transporter of RA-CH-1, B739_1343, in iron utilization and pathogenicity were investigated. Under iron-starved conditions, the mutant strain RA-CH-1ΔB739_1343 exhibited more seriously impaired growth than the wild-type strain RA-CH-1, and the expression of B739_1343 in the mutant strain restored growth. qRT-PCR results showed that the transcription of B739_1343 was not regulated by iron conditions. In an animal model, the median lethal dose (LD50) of the mutant strain RA-CH-1ΔB739_1343 increased more than 104-fold (1.6×1012 CFU) compared to that of the wild-type strain RA-CH-1 (1.43×108 CFU). In a duck co-infection model, the mutant strain RA-CH-1ΔB739_1343 was outcompeted by the wild-type RA-CH-1 in the blood, liver and brain of infected ducks, indicating that B739_1343 is a virulence factor of RA-CH-1. Finally, immunization with live bacteria of the mutant strain RA-CH-1ΔB739_1343 protected 83.33% of ducks against a high-dose (100-fold LD50) challenge with the wild-type strain RA-CH-1, suggesting that the mutant strain RA-CH-1ΔB739_1343 could be further developed as a potential live attenuated vaccine candidate for the duck industry.