Thermal treatment of inorganic thin films is a general and necessary step to facilitate crystallization and, in particular, to regulate the formation of point defects. Understanding the dependence of the defect formation mechanism on the annealing process is a critical challenge in terms of designing material synthesis approaches for obtaining the desired optoelectronic properties. Herein, we present a mechanistic understanding of the evolution of defects in emerging Sb 2 (S, Se) 3 solar cell films. We adopted a top-efficiency Sb 2 (S, Se) 3 solar-cell film in this study to consolidate our investigation. Our study reveals that the as-deposited Sb 2 (S, Se) 3 film under hydrothermal conditions generates defects with a high formation energy, demonstrating kinetically favorable defect formation characteristics. Annealing at elevated temperatures leads to a two-step defect transformation process: (1) formation of sulfur and selenium vacancy defects, followed by (2) migration of antimony ions to fill the vacancy defects. This process finally results in the generation of cation-anion anti-site defects, which exhibit low formation energy, suggesting a thermodynamically favorable defect formation feature. This study establishes a new strategy for the fundamental investigation of the evolution of deep-level defects in metal chalcogenide films, and provides guidance for designing material synthesis strategies in terms of defect control. This article is protected by copyright. All rights reserved.