Safety evaluations in preclinical studies are needed to confirm before translating a cell-based product into clinical application. We previously developed a serum-free, xeno-free, and chemically defined media (S&XFM-CD) for the derivation of clinical-grade umbilical cord-derived MSCs (UCMSCs), and demonstrated that intraperitoneal administration of UCMSCs in S&XFM-CD (UCMSC ) exhibited better therapeutic effects than UCMSCs in serum-containing media (SCM, UCMSC ). However, a comprehensive investigation of the safety of intraperitoneal UCMSC treatment should be performed before clinical applications. In this study, the toxicity, immunogenicity and biodistribution of intraperitoneally transplanted UCMSC were compared with UCMSC in rats via general vital signs, blood routine, blood biochemistry, subsets of T cells, serum cytokines, pathology of vital organs, antibody production and the expression of human-specific gene. The tumorigenicity and tumor-promoting effect of UCMSC were compared with UCMSC in nude mice. We confirmed that intraperitoneally transplanted UCMSC or UCMSC did not cause significant changes in body weight, temperature, systolic blood pressure, diastolic blood pressure, heart rate, blood routine, T lymphocyte subsets, and serum cytokines, and had no obvious histopathology change on experimental rats. UCMSC did not produce antibodies, while UCMSC had very high chance of antibody production to bovine serum albumin (80%) and apolipoprotein B-100 (60%). Furthermore, intraperitoneally injected UCMSC were less likely to be blocked by the lungs and migrated more easily to the kidneys and colon tissue than UCMSC . In addition, UCMSC or UCMSC showed no obvious tumorigenic activity. Finally, UCMSC extended the time of tumor formation of KM12SM cells, and decreased tumor incidence than that of UCMSC . Taken together, our data indicate that UCMSC display an improved safety performance and are encouraged to use in future clinical trials.