This study aims to perform multi-site calibration and validation of SWAT model using streamflow data, and investigating the responses of water balance components to land use land cover (LULC) change in the Gidabo river sub-basin of rift valley lake basin in southern part of Ethiopia. SUFI-2 algorithm embedded in the SWAT-CUP was employed for sensitivity analysis, calibration, and validation on a monthly basis. The 17 years (1990–2006) streamflow data for three (Aposto, Bedessa, and Measso) gauging stations were used for calibrating the model while 8 years (2007–2014) streamflow data was used for validating the model. The calibrated and validated SWAT model was then used to investigate the response of water balance components to LULC change for three periods (1990, 2005, and 2019) which were performed using ERDAS Imagine 2014 with a maximum likelihood classifier. The most common statistical model performance evaluation indices namely Coefficient of Determination (R2), Nash-Sutcliffe Efficiency (NSE), and Percent Bias (PBIAS) were used to evaluate performance of the SWAT model in simulating sub-basin hydrology; in addition to physical inspection of observed and simulated streamflow hydrograph. The findings of the multi-site model performance evaluation indicated that the values of R2 ranged from 0.80 to 0.64 and 0.74 to 0.72 during calibration and validation periods respectively. The values of NSE ranged from 0.74 to 0.61 and 0.71 to 0.65 during calibration and validation periods respectively whereas PBIAS ranged from 19.70 to -3.20 and 18.10 to 0.80 during calibration and validation periods respectively. The calibration and validation results indicated that the SWAT model would simulate fairly well the historical streamflow at three gauging stations. The mean annual streamflow response to LULC change for the periods 1990–2005 and 2005–2019 was observed to increase by 2.13% (1.16m3/s) and 3.62% (2.04m3/s), respectively and the mean seasonal streamflow was obtained to increase during wet season (April-September) while decreasing trend was observed during dry season (October-March) in all three gauged stations. Results also revealed that there were significant spatiotemporal variations of surface runoff, groundwater flow, lateral flow, and evapotranspiration in the sub-basin. The multi-site calibration and validation together with uncertainty analysis detects spatial variability and simulates the water balance components under changing LULC, which is paramount importance for planning and formulating appropriate integrated land and water resources management and development strategies in the rift valley lake basin of Gidabo river sub-basin in Ethiopia.