In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-β1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.