This research evaluated gender differences in the perception of safety in public transport in Santiago, Chile using quantitative and qualitative approaches. With data from the National Urban Citizen Security Survey 2019 (ENUSC), a gender comparison was made regarding the perception of safety in four scenarios: inside buses, inside the metro, at bus stops, and waiting for buses at night. Four ordinal logistic regression models were estimated to analyze how sociodemographic factors and variables associated with the perception of crime influence rider perceptions of safety in public transport. To complement the results, four focus groups were developed to obtain a deep understanding of the participants’ experiences with safety in the Santiago public transport system. We concluded that there is a high perception of insecurity in public transport for both men and women. In general, perceived insecurity inside buses, inside the metro, and waiting for public transport at night is greater among women, older people, and national citizens. Other influencing variables are the perception of insecurity regarding crime in general, the fear of being a victim of a crime, or negative situations that occur in the neighborhood, such as the presence of robberies, alcohol, and drug consumption. We proposed new variables such as fear of harassment, traffic accidents, discrimination, contagious diseases, and street protests among others. To carry out a precise public policy on this matter, a permanent scan on security issues in public transport should be developed, considering a complete set of variables. This result can be applied in Chile and all Latin American countries.