Background: Training improves dispensing behavior of drug dispensers in low- and middle-income countries. Between 2018 and 2020, a total of 5,059 Grade C pharmacists, who completed a 3-month training course for availing a “Grade C pharmacist certificate” were trained on Good Pharmacy Practice (GPP) in 11 districts in Bangladesh by Management Sciences for Health (MSH) under Better Health in Bangladesh (BHB) project. We assessed the impact of GPP among trained Grade C pharmacists under the BHB project compared to those who did not receive GPP training under the BHB project (non-trained), and explored the major challenges towards achieving GPP. Methods: We created a database of trained Grade C pharmacists provided by MSH and randomly selected the trained Grade C pharmacists for recruitment following consent. We created another database of the non-trained Grade C pharmacist who were deployed within a 1-km radius of a trained Grade C pharmacist, and randomly recruited one non-trained against one trained Grade C pharmacist. A semi structured questionnaire was administered to obtain information about knowledge of GPP, including guidelines of dispensing medicines, temperature maintenance, medicine storage, counseling customers and labeling medicines. Dispensing behavior was directly observed following a structured tool. Chi-square test (for categorical variables) and independent sample t-tests (for continuous variables) were applied for comparison between the trained and the non-trained Grade C pharmacists. A logistic regression model was applied to explore an association between knowledge and practice between the two groups. Results: Between February and March 2021, 220 trained and 220 non-trained Grade C pharmacists were recruited. Mean age (SD) of the participants was 41 years (10.5) and 98.4% were male. Compared to the non-trained, the trained Grade C pharmacists had better knowledge about the guidelines of dispensing medicines (97.7% vs 89.5%, p < 0.001), temperature maintenance (91.8% vs 45.5%, p = 0.001), medicine storage (92.3% vs 40.5%, p = 0.001) counseling customers (99.5% vs 92.3%, p <.001) and labeling medicines (91.0% vs 80%, p < 0.001). General dispensing behavior was observed to be better among the trained than the non-trained with labeling of medicines (63.2% vs 53.4%, p = 0.038), counseling customers (39.1% vs 28.6%, p = 0.021) and using a room thermometer for maintaining ambient temperature in the medicine shops (56.8% vs26.8%, p < 0.001). Bad behavior of the customers (39.5%) and lack of GPP knowledge among Grade C pharmacists (28.6%) were recognized to be challenges towards achieving GPP in Bangladesh. Conclusion: Training led to better knowledge and practices about dispensing medicines among Grade C pharmacists in Bangladesh. Periodic training may promote achieving GPP in Bangladesh.