Osteocytes play a key role in orchestrating bone homeostasis and turnover and, therefore, in vitro investigations with osteocytes are of high relevance for biomaterial and drug testing in future. In this study, collagen type I gels and collagen gels modified with biomimetically mineralized collagen were tested as three-dimensional (3D) environment for the maintenance of the osteocytic phenotype of primary human osteocytes. After cultivation in different collagen gels, cells were analyzed microscopically for the osteocytic phenotype and gene expression of osteocyte marker genes osteocalcin, podoplanin (PDPN)/E11, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP-1), and sclerostin (SOST). Directly after isolation from bone tissue the cells expressed all examined osteocyte markers. After 7 days of 3D cultivation in collagen gels the osteocytic marker genes MEPE and SOST were upregulated and the other marker genes still expressed. Modification of collagen gels with biomimetically mineralized collagen and strontium-doped mineralized collagen prevented the cell-seeded gels from shrinking. Osteocyte morphology was not affected by the gel modification. However, the isolation of RNA from the mineralized gel variants was heavily impaired. Alternatively, the osteocytic differentiation of human osteoblasts in the different collagen gels was examined. Primary human osteoblasts were embedded into the gels and cultivated under osteogenic stimulation. After 14 days of cultivation, embedded osteoblasts showed osteocyte-like morphology and positive staining for DMP-1. Early osteocyte marker genes, such as PDPN/E11 and PHEX, were expressed while the expression of the osteoblast marker gene alkaline phosphatase (ALPL) increased. This ALPL upregulation was partly prevented by modification of collagen gels with mineralized collagen. This research focuses on the in vitro three-dimensional cultivation of primary human osteocytes instead of rodent osteocyte cell lines. Stable in vitro cultures of these regulating cells provide the opportunity to establish co- and triple cultures with osteoblasts and osteoclasts to analyze the cross talk between these cell species and to establish in vitro bone models for the testing of bioactive molecules, growth factors, drugs, and biomaterials.