Osteoclasts are pivotal cells for bone remodeling and their activity is coordinated by osteocytes that reside inside the bone matrix. In vitro co-cultures of osteocytes and osteoclasts are therefore advantageous to analyze the crosstalk between these cell species. In this study, primary osteocytes were isolated from human bone in a multistep isolation process and embedded into three-dimensional collagen gels. Mature human osteoclasts were generated by differentiation of human peripheral blood mononuclear cells (PBMCs). Different surfaces were tested for osteoclast formation: suspension dishes, collagen gels, and normal tissue culture polystyrene. After detachment from the surfaces, osteoclasts showed typical morphology and gene expression of osteoclast markers. Osteoclasts that were differentiated on collagen exhibited the highest osteoclast marker expression. Cocultivation of mature osteoclasts with osteocytes was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoclasts on the basal side of a porous polyethylen terephtalate membrane, which allowed the separate gene expression analysis for osteocytes and osteoclasts. After 7 days of co-culture both cell species showed their typical morphology, which is multinucleated giant cells for osteoclasts and star-shaped cells with dendritic extensions for osteocytes. Furthermore, osteoclast markers tartrate-resistant acid phosphatase, carbonic anhydrase II, and cathepsin K were detected both on gene expression and protein level in single and co-cultures. Osteocytes showed gene expression of typical osteocyte markers E11, sclerostin, dentin matrix protein 1, osteocalcin, and receptor activator of nuclear factor-κ ligand both in single and co-culture. This study is the first to establish an in vitro bone model that contains both primary human osteocytes and primary human osteoclasts. Previous studies applied rodent osteocyte cell lines to examine the influence of osteocytes on osteoclast function. This model mimics the clinical situation better since osteocytes are postmitotic cells whose function might be different in primary state compared with a proliferating cell line. Furthermore, the co-culture model can be the basis for in vitro triple culture models involving osteoblasts as the third bone cell species.