The present study aims to evaluate plastic deformation's effect by cold-rolling on the precipitation sequence of 2024 aluminum alloy. X-ray diffraction, scanning/transmission electron microscopy, and Vickers microhardness tests have been used to characterize the microstructure and mechanical behavior of the alloys. It was observed that plastic deformation induces changes in the precipitation sequence, which affects the mechanical properties and delays the overaging stage. In the deformed alloy, two hardening peaks were observed. These peaks occurred at 30 min (248 HV ± 5) and 600 min (230 HV ± 2) and were attributed to the θ' and S' phases, respectively. However, in the non-deformed alloy, only a single hardening peak was observed. This peak arose after 300 min aging (208 HV ± 4) and was attributed to the S' phase formation. Thus, the precipitation sequence in the deformed alloy was the following: αSSS–CuMg clusters → GPB-II zones/θ''/θ'/ → S' → S, and for the non-deformed alloy was αSSS–CuMg clusters → GPB-II zones/S'/ → S.