To gain more insight into the impact of zinc on the primary metabolites in rapeseed, plants were regenerated in vitro in the presence of zinc (0.1–1 mM), acclimatized, transferred to a greenhouse, and treated with 2 mM ZnSO4. The main metabolites, including soluble carbohydrates and free amino acids, were analyzed by nuclear magnetic resonance spectroscopy, and further confirmed by spectrophotometry and enzymatic analyses. Exposure of these greenhouse-grown plants to ZnSO4 led to both a significant rise of the total amino acid level and an increase of proline accumulation in the different tissues. Moreover, the control plants, regenerated without zinc stress, exhibited a significant reduction of soluble carbohydrate levels, whereas the plants derived from Zn-treated regenerants exhibited significantly higher levels of both glucose and sucrose. These levels increased proportionally with the Zn concentration in the regeneration step.