The dechlorination of polychlorinated biphenyl (PCB) has been studied intensively, while the comprehensive understanding of the electron transfer and selectivity pattern mechanism is still limited. Therefore, in this study, the reactivity of nickel/zero-valent iron (Ni/Fe) was investigated during the dechlorination of 2,2′,4,4′,5-pentachlorobiphenyl (PCB-99) at various temperatures. Gas chromatography-mass spectrometry analysis and quantum chemical calculations, including bond dissociation energy, C-Cl bond length, and the lowest unoccupied molecular orbital (LUMO), were used to explore the dechlorination pathways and the electron selectivity. Results showed that the reduction efficiency of PCB-99 (100 μg L⁻¹) did not increase monotonically with the temperature, but followed the order: 25 °C (75.9%) > 15 °C (62.5%) > 35 °C (51.6%), due to the thermodynamic evolution of Fe species to generate more Fe(II) and the abundance of oxygen vacancies at 25 °C. However, as the temperature continued raising to 35 °C, Ni/Fe was oxidized heavily and aggregated into large particles, significantly reducing the electron density of Fe(II) and inhibiting the electron transfer. The main dechlorination pathway was PCB-99 → PCB-47 → PCB-17 → PCB-8 → PCB-3 → biphenyl, indicating that electrons tended to attack C-Cl bonds following the order meta- > para- > ortho-position, which could be better explained by LUMO. Overall, this work is expected to improve the understanding of the dechlorination behavior of PCB-99 from both fundamental and applied perspectives.