Accurate soil organic carbon models are key to understand the mechanisms governing carbon sequestration in soil and to help develop targeted management strategies to carbon budget. The accuracy and reliability of soil organic carbon (SOC) models remains strongly limited by incorrect initialization of the conceptual kinetic pools and lack of stringent model evaluation using time-series datasets. Notably, due to legacy effects of management and land use change, the traditional spin-up approach for initial allocation of SOC among kinetic pools can bring substantial uncertainties in predicting the evolution of SOC stocks. The AMG model can fulfill these conditions as it is a parsimonious yet accurate SOC model using widely-available input data. In this study, we first evaluated the performance of AMGv2 before and after optimizing the potential mineralization rate (k0) of SOC stock following a leave-one-site-out cross-validation based on 24 long-term field experiments (LTEs) in the Southwest of China. Then, we used Rock-Eval® thermal analysis results as input variables in the PARTYSOC machine learning model to estimate the initial stable SOC fraction (CS/C0) for the 14 LTEs where soil samples were available. The results showed that initializing the CS/C0 ratio using PARTYSOC combined with the optimized k0 further improved the accuracy of model simulations (R2 = 0.87, RMSE = 0.25, d = 0.90). Combining average measured CS/C0 and k0 optimization across all 24 LTEs also improved the model predictive capability by 25% compared to using default parameterization, thus suggesting promising avenue for upscaling model applications at the regional level where only a few measurement data on SOC stability can be available. In conclusion, the new version of the AMG model developed in the Tuojiang River Basin context exhibits excellent performance. This result paves the way for further calibration and validation of the AMG model in a wider set of contexts, with the potential to significantly improve confidence in SOC predictions in croplands over regional scales.