This study investigated the plastic deformation behavior of a polycrystalline L12-structured FeCoNiV high-entropy alloy (HEA) using in situ electron backscatter diffraction (EBSD) and digital image correlation (DIC) methods. The different deformation mechanisms in two HEAs, which affected their mechanical performance, were explored using a face-centered cubic (FCC)-structured sample for comparison. Using slip traces and lattice rotation path analysis, {111}<110> slip systems were found to be activated in the L12-structured FeCoNiV HEA. In addition, a lower average lattice rotation rate was estimated for this sample compared to that of the FCC specimen; this macroscopically verified the existence of additional obstacles to dislocation slip caused by the ordered structure during plastic deformation, and was found to contribute to the high strength of the L12-structured FeCoNiV HEA. Furthermore, these additional obstacles blocked the formation of deformed substructures in the L12-structured sample and aggravated the intergranular incompatibility, which enabled crack initiation at the grain boundaries. These findings are important for understanding the deformation behavior and fracture mechanism in L12-structured HEAs and for designing new high-performance ordered HEAs.