Introduction: The knowledge loss, especially in medicine has long been an issue of concern for all health care providers and is poorly investigated throughout the world. In this study we focused on pharmacological knowledge in Ahvaz Jundishapur University of Medical Sciences in Iran. Methods: 110 volunteer of medical students who had previously passed two partial basic pharmacology exams were retested (an unscheduled retention test) again in three group, which were in the 9th (n=44), 12th (n=37), and 15th (n=29) semester of their education to be evaluated for loss of their pharmacological knowledge. Results: The average score of partial exams in all three groups was significantly higher than the score of retention test (12.74±0.31 vs. 9.93±0.44, 15.85±0.35 vs. 12.57±0.43, and 13.31±0.35 vs. 11.68±0.61 of the students spending 9th, 12th, and 15th semesters, respectively). The amount of knowledge loss was 22±0.6, 20±0.7, and 12±0.75% of the students spending 9th, 12th, and 15th semesters, respectively. There was a significant decrease in the value of knowledge loss of the students spending the15th semester in comparison to the other groups (p<0.05). Conclusion: These results showed that when the time period between the first exposure to pharmacological education and retention test, is increased, the amount of knowledge loss becomes smaller. The reason may be due to more practical rehearsal of pharmacology in clinical years.