In recent years, the Yangtze River Economic Belt has gradually built an integrated transportation system. Under this context, the dynamic cooperative development of waterway and railway is measured by the natural experiment of newly built freight railway in port cities along the upper reaches of the Yangtze River. A difference-in-difference model is adopted to estimate the impact of this railway on water transport in the surrounding area. It is found that instead of substitute the water transport, the railway actually promotes the growth of waterway freight volume, therefore, the complementation effect is more obvious. The results expand the empirical understanding of the interactive relationship between waterway and railway transportation in the upper reaches of the Yangtze River and provide important policy implications.