Clinical applications of mesenchymal stem cells (MSCs) rely on their capacity to home and engraft in the appropriate target injury tissues for the long term. However, their homing efficiency has been observed to be very poor because of the lack or modifications of homing factors SDF‐1α and CXCR4 receptors. Hence, this study was designed to investigate the homing and retention of pretreated human adipose tissue‐derived MSCs (hASCs) from three different delivery routes in response to SDF‐1α, released from chitosan‐based injectable hydrogels. After stimulation of ASCs with a hypoxia mimicking agent, the expression level and functionality of CXCR4 were analyzed by flowcytometric analysis (FACS), transwell migration assay and qPCR. Then, the homing/retention of pretreated DiI‐labeled hASCs were compared through three different in vivo delivery routes, 2 weeks after transplantation in Wistar rats. The cells were tracked histologically by fluorescent microscope and by PCR for human‐specific CXCR4 gene. Results showed CXCR4 has dynamic expression pattern and pretreatment of hASCs significantly up‐regulates CXCR4, leading to an increase in migration capacity toward 100 ng/mL SDF‐1α in vitro and homing into the subcutaneously implanted hydrogel releasing SDF‐1α in vivo. Furthermore, it seems that SDF‐1α is particularly important in the retention of ASCs, in addition to its chemoattraction role. In summary, the delivery route in which the ASCs were mixed with the hydrogel rather than systemic delivery and local injection and preconditioning undertaken to increase CXCR4 expression concomitant with SDF‐1α delivery by the injectable hydrogel, allowed for further homing/retention of ASCs. This might be a promising way to get better therapeutic outcomes in stem cell therapy.