Pilates is an effective exercise method for rehabilitating musculoskeletal disorders as its principles are based on the activation of local muscles. This study aimed to compare the subjects with and without Pilates experience to find out the effect of the experience on the core muscle activity and muscle co-contraction, and to examine the relationship between the core muscle activation level and the kinematic data. This study involved 32 subjects, including 16 experienced Pilates practitioners and 16 non-experienced subjects. The knee stretch on the reformer was performed in three different positions: flat back with a neutral pelvis, round back with posteriorly tilted pelvis (RPP), and extended back anteriorly tilted pelvis (EAP). The electromyography of the internal oblique (IO), rectus abdominis (RA), multifidus (MU), and iliocostalis lumborum (IL) muscles were measured, as well as kinematic data from a 3D motion analysis system. Compared to the non-experienced subjects, the experienced subjects activated the IO muscles more than the RA muscles, and the most significant difference was seen in the RPP position ( < 0.05). The experienced patients activated the MU muscles more often than the IL muscles, with the most significant difference observed in the RPP position and the least significant in the EAP position ( < 0.05). All kinematic data and muscle activity (IO, IO/RA ratio, MU/IL ratio) showed significant differences between the experienced and non-experienced subjects ( < 0.05). The subjects presented a moderate correlation between muscle activation and core stability. It was confirmed that the experienced Pilates practitioners activated the abdominal and low back core muscles effectively, and the stability of the pelvis and trunk were better than that of the non-experienced participants. In addition, the better the trunk stability was maintained, the larger and more accurate movement of the mobility segment was observed.