Mesenchymal stem cells (MSCs) have generated tremendous interest for treating various diseases due to their self-renewal and differentiation capacities. Many studies have demonstrated the immunoregulatory capability of MSCs; however, most of these studies were conducted with fetal bovine serum (FBS), which has an uncertain composition. In this study, we established a serum-free, xeno-free, completely chemically defined medium for the proliferation and expansion of rhesus monkey bone marrow (BM)-derived MSCs (rBMSCs) in vitro. The growth kinetics, characteristics, immunophenotype, and immunosuppressive abilities of rBMSCs grown in serum-free media (SFM) were evaluated and compared with those of cells grown in serum-containing media (SCM). Moreover, we employed RNA sequencing to evaluate the expression pattern of genes related to immune responses in both culture conditions. Compared to cells grown in SCM, rBMSCs grown in SFM exhibited better biological characteristics regarding cell proliferation and immunosuppressive abilities. Cells from both media types exhibited similar immunophenotypic expression patterns for CD29, CD34, CD45, HLA-DR, CD73, CD90, and CD105. Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and Gene Set Enrichment Analysis (GSEA) revealed that CXCL8 was downregulated by 4.1 fold in SFM-cultured rBMSCs compared with those in SCM. Furthermore, the mixed lymphocyte culture revealed that the proliferation activity and the expression levels of inflammatory factors of peripheral blood mononuclear cells (PBMCs) were significantly decreased after the addition of the CXCL8 neutralizing antibody, which was related to the elevated immunosuppressive abilities of SFM-suspended rBMSCs. These results suggest a possible cell culture method as well as immunoregulatory mechanisms for clinical cell therapies requiring nonanimal-derived components.