Mesenchymal stem/stromal cells (MSCs) have been studied intensively due to their great potential in regenerative medicine. Before therapeutic application, in vitro expansion of MSCs is normally required to achieve a sufficient number of cells. However, previous studies have indicated that different expansion procedures may result in an alteration of MSC growth kinetics and their biological properties, such as microRNA (miRNA) expression. This study aimed to assess the effects of culture conditions on adipose-derived MSCs (ASCs) behaviours during long-term in vitro expansion. Three different mediums, including the traditional DMEM supplemented with 10% fetal bovine serum (FBS), a commercially reduced serum medium, and a xeno-free medium were used for ASC culture. The changes in cell morphology, population doubling time, cell proliferation, intracellular reactive oxygen species (ROS) level, and expression levels of miR-21, miR-223, and miR-423 of cultured ASCs were analyzed at passage (P) 7, P9, and P11. A gradual decrease in proliferation rate and prolonged population doubling time was observed in ASCs cultured in reduced serum and xeno-free mediums over time, whereas the cells rapidly underwent cellular senescence and stopped proliferating at P9 in DMEM supplemented with 10% FBS. The levels of ROS varied greatly depending on particular culture conditions. A reduction of miR-21 and miR-223 expression was detected at the later passages, whereas the miR-423 level remained unaffected under xeno-free condition. Our study indicated that an appropriate selection of culture condition is important to retain the desired biological properties of ASCs in order to utilize their potential in therapeutic applications.