The role of mesenchymal stem cells (MSC) in osteosarcoma (OS), the most common primary tumor of bone, has not been extensively elucidated. We have recently shown that OS is characterized by interstitial acidosis, a microenvironmental condition that is similar to a wound setting, in which mesenchymal reactive cells are activated to release mitogenic and chemotactic factors. We therefore intended to test the hypothesis that, in OS, acid‐activated MSC influence tumor cell behavior. Conditioned media or co‐culture with normal MSC previously incubated with short‐term acidosis (pH 6.8 for 10 hr, H⁺‐MSC) enhanced OS clonogenicity and invasion. This effect was mediated by NF‐κB pathway activation. In fact, deep‐sequencing analysis, confirmed by Real‐Time PCR and ELISA, demonstrated that H⁺‐MSC differentially induced a tissue remodeling phenotype with increased expression of RelA, RelB and NF‐κB1, and downstream, of CSF2/GM‐CSF, CSF3/G‐CSF and BMP2 colony‐promoting factors, and of chemokines (CCL5, CXCL5 and CXCL1), and cytokines (IL6 and IL8), with an increased expression of CXCR4. An increased expression of IL6 and IL8 were found only in normal stromal cells, but not in OS cells, and this was confirmed in tumor‐associated stromal cells isolated from OS tissue. Finally, H⁺‐MSC conditioned medium differentially promoted OS stemness (sarcosphere number, stem‐associated gene expression), and chemoresistance also via IL6 secretion. Our data support the hypothesis that the acidic OS microenvironment is a key factor for MSC activation, in turn promoting the secretion of paracrine factors that influence tumor behavior, a mechanism that holds the potential for future therapeutic interventions aimed to target OS.