In 2012, Colombia implemented a school-based HPV vaccination program of a 3-dose series for nine year old girls. Following a mass psychogenic response after vaccination in a Colombian town, vaccination rates dropped from 80% in 2012–2013 to 5% in 2016. The study aimed to identify barriers and facilitators of HPV vaccine uptake among girls eligible for vaccination in the initial years of vaccine implementation from 2012 to 2014, and their parents. We conducted 19 individual qualitative interviews and 18 focus groups with an average of 5 girls, in Manizales, Colombia between 2016 and 2017. In total, 49 girls from six schools and 58 of their parents participated in the study. Participants had some degree of awareness about cervical cancer, especially among those of middle and upper socioeconomic level. However, the vaccine was known as a prevention measure only after pap-smears and condoms. The main facilitator for vaccine uptake for parents was the desire to prevent diseases in general and for girls, it was facilitated by receiving positive information about the vaccine. The main barriers for vaccine uptake or for three doses completion were the event in Carmen de Bolivar, fear of adverse effects and fear of needles. Girls and parents stated that they received little or no information from schools or health care services about the HPV vaccine prior to vaccination. Our results suggest that improving HPV vaccination rates in Colombia will require a comprehensive education program including mass media information about HPV vaccine.