We created the "3-dimensional synaptic puzzle" (3Dsp) as an educational resource for the physiology teaching of synaptic transmission (ST). In this study, we aimed to apply and evaluate the use of 3Dsp. For this, we divided 175 university students from public and private universities into two groups: (1) control (CT; students that were only exposed to traditional class or video lessons about ST), and; (2) test (3Dsp; students that were exposed to the 3Dsp practical class in addition to the traditional theoretical class). ST knowledge of students was evaluated before, immediately after, and 15 days after interventions. Additionally, students completed a questionnaire about their perception of teaching-learning methods used in physiology classes and their self-perception of engagement in the physiology content. The CT groups improved their ST knowledge score from pretest to immediate (P < 0.0001 for all groups) and late posttest (P < 0.0001 for all groups). 3Dsp groups also enhanced their score from pretest to immediate (P = 0.029 for public university students; P < 0.0001 for private university students) and late posttest (P < 0.0001 for all groups). We also observed improvement from the immediate to late posttest in the 3Dsp group from private universities (P < 0.001). Both private groups performed better in general ST and specific electrical synapse questions in the pretest and immediate posttest compared to the public CT group (P < 0.05 for all comparisons). More than 90% of the students from both universities affirmed that the 3Dsp contributed to their physiology comprehension and that they would recommend the use of the 3-D models to other teachers in their classes.