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The cell–biomaterial interface is highly complex; thousands of molecules and many processes participate in its formation. Growing demand for improved biomaterials has highlighted the need to understand the structure and functions of this interface. Proteomic methods offer a viable alternative to the traditional in vitro techniques for analyzing such systems. Magnesium is a promoter of cell adhesion and osteogenesis. Here, we used the LC-MS/MS to compare the protein expression profiles of human osteoblasts (HOb) exposed to sol-gel coatings without (MT) and with Mg (MT1.5Mg) for 1, 3, and 7 days. PANTHER, DAVID, and IPA databases were employed for protein identification and data analysis. Confocal microscopy and gene expression analysis were used for further characterization. Exposure to MT1.5Mg increased the HOb cell area and the expression of SP7, RUNX2, IBP3, COL3A1, MXRA8, and FBN1 genes. Proteomic analysis showed that MT1.5Mg affected the early osteoblast maturation (PI3/AKT, mTOR, ERK/MAPK), insulin metabolism, cell adhesion (integrin, FAK, actin cytoskeleton regulation) and oxidative stress pathways. Thus, the effects of Mg on cell adhesion and osteogenesis are rather complex, affecting several pathways rather than single processes. Our analysis also confirms the potential of proteomics in biomaterial characterization, showing a good correlation with in vitro results. ; Funding for open access charge: CRUE-Universitat Jaume I ; This work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21]; Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069]; Universitat Jaume I [UJI-B2021-25]; and Basque Government [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, José Ortega, José Miguel Pedra and Iraide Escobés for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for making the titanium discs.