The aim of this study was to evaluate the effect of three training methodologies on the acquisition of psychomotor skills for laparoendoscopic single-site surgery (LESS), using straight and articulating instruments. A prospective study was conducted with subjects randomly divided into three groups, who performed a specific training for 12 days using three laparoscopic tasks in a laparoscopic simulator. Group-A trained in conventional laparoscopy setting using straight instruments and in LESS setting using both straight and articulating instruments. Group-B trained in LESS setting using straight and articulating instruments, whereas Group-C trained in LESS setting using articulating instruments. Participants' performance was recorded with a video-tracking system and evaluated with 12 motion analysis parameters (MAPs). All groups obtained significant differences in their performance in most of the MAPs. Group-C showed an improvement in nine MAPs, with a high level of technical competence. Group-A presented a marked improvement in bimanual dexterity skills. Training in LESS surgery using articulating laparoscopic instruments improves the quality of skills and allows smoother learning curves.