This was a quasi-experimental study proposed to assess knowledge and attitude towards human papillomavirus (HPV), cervical cancer, and HPV vaccine. This study also aimed to determine acceptability of the HPV vaccine, and to compare knowledge and attitude towards HPV, cervical cancer, and HPV vaccine, and acceptability of HPV vaccine between before and after intervention among students attending colleges of public health in southern Thailand. A total of 322 samples were recruited from the first year students of certificate program in public health, 176 was the intervention group and 156 was the control group. Data was analyzed using frequency, percentage, mean, standard deviation, chi-square test, paired t-test, and McNemar test. The results revealed that knowledge of the intervention group increased significantly after intervention (p < 0.05). Before intervention, 54.5% of the intervention group acknowledged on HPV, cervical cancer, and HPV vaccine at moderate level and 61.4% improved knowledge to high level after intervention. On the other hand, knowledge of the control group was observed at moderate level before and after intervention, 50.3% and 52.6%, respectively. Proportion of students who had positive attitude in the intervention group rose from 66.5% to 86.4% after intervention. However, proportion of students who had positive attitude in the control group was not different between before and after intervention (p > 0.05). Willingness to receive HPV vaccine in the intervention group rose from 55.7% to 69.4% after intervention whereas there was a decrease from 80.1% to 68.6% in the control group. This study concluded that educational tools affected adolescents to improve their knowledge and attitude towards HPV, cervical cancer, and HPV vaccine, and acceptability of HPV vaccine.