Purpose Cervical cancer is an important cause of mortality in low- and middle-income countries. Although screening technologies continue to improve, systems of care remain fragmented. It is important to better understand factors that affect use of screening services and loss to follow-up along the care continuum. Methods We conducted a mixed-methods study of a cytology-based screening program in rural Guatemala. A retrospective electronic chart review was performed on data from all patients from 2013 to 2014. We analyzed progression through care and calculated loss-to-follow-up rates. We also analyzed the prior experiences of patients with cervical cancer screening on the basis of self-reported historical data available in the chart review. Structured interviews with a subset of individuals to explore social supports and barriers to screening and engagement in care were conducted at the time of screening. Results The analysis included 515 women (median age, 36 years). Cytologic screening showed concern for neoplastic changes in 0.83%; half resulted in biopsy-proven cervical intraepithelial neoplasia. An additional 9.9% showed severe inflammation. The rate of loss to follow-up was 11.3%. All losses to follow-up occurred for severe inflammation, not for cervical intraepithelial neoplasia. Historical data showed that 73% of the cohort had previously been screened and had high levels of loss to follow-up (57.4%). Qualitative interviews revealed factors that promoted loss to follow-up; these included cost, lack of social supports, transportation, distrust in public facilities, long turn-around times, and failure to return test results or offer follow-up treatments. Conclusions Taken together, these quantitative and qualitative results highlight the need for cervical cancer screening programs in Guatemala to improve uptake of screening services by eligible women and to improve follow-up after a first abnormal screen.