Establishment of a three-dimensional (3-D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four-point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60–88 μm), type II (88–100 μm) and type III (100–125 μm). To establish the 3-D mechanical loading system, PLGA membrane was integrated with conventional force-loading plates and the third passage skeletal myoblasts from neonatal Sprague–Dawley (SD) rats were seeded. Small PLGA membranes were put in 24-well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days’ culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 μstrain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3-D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four-point bending strain unit provides a satisfactory 3-D mechanical loading system.