The natural flow regime within a watershed can be considered as the expected temporal patterns of streamflow variation in the absence of human impacts. While ecosystems have evolved to function under these conditions, the natural flow regime of most rivers has been significantly altered by human activities. Land use change, including the development of agriculture and urbanization, is a primary cause of the loss of natural flow regimes. These changes have altered discharge volume, timing, and variability, and consequently affected the structure and functioning of river ecosystems. The Meramec River watershed is located in east central Missouri and changes in land use have been the primary factor impacting flow regimes across the watershed. In this study, a watershed model, the Soil and Water Assessment Tool (SWAT), was developed to simulate a long-term time series of streamflow (1978-2014) within the watershed. Model performance was evaluated using statistical metrics and graphical technique including R-squared, Nash-Sutcliffe efficiency, cumulative error, and 1:1-ratio comparison between observed and simulated variables. The calibrated and validated SWAT model was then used to quantify the responses of the watershed when it was a forested natural landscape. An Indicator of Hydrologic Alteration (IHA) approach was applied to characterize the flow regime under the current landcover conditions as well as the simulated natural flow regime under the no land use change scenario. Differences in intra- and inter-annual ecologically relevant flow metrics were then compared using SWAT model outputs in conjunction with the IHA approach based on model outputs from current and no land use change conditions. This study provides a watershed-scale understanding of effects of land use change on a river’s flow variability and provides a framework for the development of restoration plans for heavily altered watersheds.