Electron backscatter diffraction (EBSD) coupled with in-situ tensile loading is powerful for investigating the microstructural evolution of alloys. Thermo-mechanically treated f.c.c. medium-entropy alloys (MEAs) typically have high densities of annealing twin boundaries (ATBs), which can not only strengthen but also toughen the MEA via interacting with dislocations. However, the evolution of ATBs and other substructures in plastically-deformed MEA has not yet been revealed. Plastic deformation involving dislocation evolution, active slip systems, lattice rotation, boundary transformation, and grain subdivision in a polycrystalline MEA Ni41.4Co23.3Cr23.3Al3Ti3V6 was studied using in-situ EBSD. The slip was accompanied by heterogeneous lattice rotation among grains and within grains, where inhomogeneous plasticity was accommodated by geometrically-necessary dislocations (GNDs). Both GND and low-angled boundaries (LABs) densities substantially increased with progressive strain, which was mainly concentrated in sites approaching ATBs or grain boundaries (GBs). Located stress, lattice rotation, or curvature caused a loss in the coherence of ATBs, which resulted in integrity loss with increasing strain and promoted a decrease in density by 60 %. Further, lattice rotation incompatibility due to constraints from neighboring grains leads to grain fragmentation into various misorientated volumes, which were separated by LABs or high-angle boundaries (HABs). The grain orientation angle increased with progressive strain and crystallographic 〈111〉 orientation gradually spread toward a tensile direction. Slip systems with maximum Schmid factor were activated first at ε ≥ 3.9 %, which is almost the same with experimental slip traces. Both single slip and double slip occurred during plasticity, where straight slip traces tend to curve due to lattice curvature. Slip transfers are not only controlled by geometric compatibility factor, which can occur between some neighboring grains with low geometric compatibility factor but high Schmid factor.