The study aimed to investigate the effect of a training program based on the principles of conceptual understanding in enhancing the pedagogical knowledge of mathematics teachers. The study sample consisted of 34 male and female in-service mathematics teachers, who teach the middle grades (5-8). To achieve the objectives of the study, a quasi-experimental approach was used, with a pre-post design for two groups. In order to collect data, a pedagogical knowledge test was used for the content of numbers and algebra contained in the middle grades curriculum. The results showed a significant effect of the training program in enhancing the pedagogical knowledge of mathematics teachers, with two dimensions (knowledge of teaching mathematics: approaches and strategies, knowledge of students’ thinking). The results also showed an improvement in performance levels on the test in general among the teachers of the experimental group, as the percentage of teachers in the level of skilled performance increased from 0% on the pre-test to 53% on the post-test. In light of the results, the study recommended to conduct more research through employing more principles of conceptual understanding to enhance pedagogical knowledge among broader samples of mathematics teachers and different mathematics content.