Prolonged soil cultivation has been identified as a major cause of land degradation and a threat to soil quality in drought-prone environments. This study evaluated land use effects on soil carbon (C) fractions and organic C (SOC) quality across three semiarid agro-ecosystems (Harrismith, Tweespruit, and Kroonstad) in central South Africa. Soil samples were collected from croplands, primary and secondary grasslands at the 0- to 200-mm layer in each agro-ecosystem and analyzed for various soil C fractions. The SOC structure was characterized with C-13 nuclear magnetic resonance (NMR) spectroscopy. All analyses were done on bulk soil samples. Conversion of primary grasslands into croplands decreased C fractions by 27 to 90% across the three agro-ecosystems, with highest losses recorded in Harrismith and Tweespruit and lowest in Kroonstad, suggesting site-specific conditions acted together with cultivation to effect C losses. The C-13 NMR spectra revealed a slight change in SOC structural composition when O-alkyl C decreased with concomitant increase in aromatic and alkyl C due to cultivation, with differences in the range of 1 to 11%. O-alkyl C remained almost the same in Harrismith, suggesting that lignin-derived methoxy groups were probably more dominant than easily decomposable carbohydrates as opposed to O-alkyl C in Tweespruit and Kroonstad. Meanwhile, reversion of cultivated soils into perennial pastures restored and even increased some soil C fractions (by 3-129%) to represent primary grasslands, especially in Kroonstad. Organic C decomposition was lower in the cultivated soils relative to virgin and restored soils. This underscores the importance of determining plant biomass composition because these unusual responses were probably related to vegetation differences. Overall, this study demonstrates the potential of secondary grassland management to rehabilitate degraded cultivated soils with implications for restoration of agro-ecosystem functions and services.