Stacking fault energy (SFE) plays an important role in deformation mechanisms and mechanical properties of face-centered cubic (fcc) metals and alloys. In many concentrated fcc alloys, the SFEs determined from density functional theory (DFT) calculations and experimental methods are found having opposite signs. Here, we show that the negative SFE by DFT reflects the thermodynamic instability of the fcc phase relative to the hexagonal close-packed one; while the experimentally determined SFEs are restricted to be positive by the models behind the indirect measurements. We argue that the common models underlying the experimental measurements of SFE fail in metastable alloys. In various concentrated solid solutions, we demonstrate that the SFEs obtained by DFT calculations correlate well with the primary deformation mechanisms observed experimentally, showing a better resolution than the experimentally measured SFEs. Furthermore, we believe that the negative SFE is important for understanding the abnormal behaviors of partial dislocations in metastable alloys under deformation. The present work advances the fundamental understanding of SFE and its relation to plastic deformations, and sheds light on future alloy design by physical metallurgy. Display omitted •The common models underlying experimental measurements of stacking fault energy fail in metastable alloys.•Theoretical stacking fault energy correlates with the Gibbs free energy difference between the fcc and hcp phases.•Ab initio calculated stacking fault energy correlates nicely with the prevailing deformation mechanism.•Negative stacking fault energy plays critical role in understanding dislocation behaviors in metastable alloys.