Background: Cadmium (Cd), a ubiquitous and highly toxic heavy metal pollutant, is toxic to animals and plants. Calcium (Ca) is an essential component for plant growth and reduces plant Cd adsorption by competing with Cd. To gain deeper insight into the effects of Ca on Cd absorption, translocation, subcellular distribution, and chemical forms in S. matsudana seedlings under Cd stress, an investigation was conducted on these properties. Results: Adding Ca alleviated Cd physiological toxicity in S. matsudana, reduced Cd adsorption, increased the translocation from roots to shoots, lead to subcellular redistribution of Cd by increasing the proportion of Cd in soluble fractions but decreasing Cd in the cell wall and changed the chemical forms of Cd from 0.6 M HCl- and 2% HAc-extracted Cd to 1 M NaCl-extracted Cd. The energy dispersive X-ray analyses (EDXA) results revealed that after adding Ca, Cd was transferred through the root epidermis, cortex, endodermis, and vascular cylinder, transported to the shoots, and was highly accumulated in leaf epidermal and mesophyll cells, but less in leaf vein and guard cells. The genes involved in Cd uptake and xylem loading included NRAMP1, ZIP8, HMA2, and HMA4, which were up-regulated significantly (p < 0.05) in the Cd and Cd + Ca treatments compared to the control. Conclusions: The findings of this study provide new insight into the mechanism that Ca alleviates Cd toxicity in woody tree species, as well as propose an important prospect of Ca addition for improving the phytoremediation of Cd contamination.