Terrestrial ecosystems in the southern United States (SUS) have experienced a complex set of changes in climate, atmospheric CO2 concentration, tropospheric ozone (O3), nitrogen (N) deposition, and land-use and land-cover change (LULCC) during the past century. Although each of these factors has received attention for its alterations on ecosystem carbon (C) dynamics, their combined effects and relative contributions are still not well understood. By using the Dynamic Land Ecosystem Model (DLEM) in combination with spatially explicit, long-term historical data series on multiple environmental factors, we examined the century-scale responses of ecosystem C storage and flux to multiple environmental changes in the SUS. The results indicated that multiple environmental changes shifted SUS ecosystems from a C source of 1.20 ± 0.56 Pg (1 Pg = 1015 g) during the period 1895 to 1950, to a C sink of 2.00 ± 0.94 Pg during the period 1951 to 2007. Over the entire period spanning 1895–2007, SUS ecosystems were a net C sink of 0.80 ± 0.38 Pg. The C sink was primarily due to an increase in the vegetation C pool, whereas the soil C pool decreased during the study period. The spatiotemporal changes of C storage were caused by changes in multiple environmental factors. Among the five factors examined (climate, LULCC, N deposition, atmospheric CO2, and tropospheric O3), elevated atmospheric CO2 concentration was the largest contributor to C sequestration, followed by N deposition. LULCC, climate, and tropospheric O3 concentration contributed to C losses during the study period. The SUS ecosystem C sink was largely the result of interactive effects among multiple environmental factors, particularly atmospheric N input and atmospheric CO2.