Background Anal cancer rates are increasing in HIV-infected men. Screening programmes similar to prostate and cervical cancer have been recommended to reduce morbidity and mortality. Research shows that screening processes have psychological consequences that need to be considered. Limited investigation of the psychological impact of anal cancer screening has been conducted. Methods A prospective longitudinal survey of 291 men was conducted at three time points over 14 weeks at a public HIV clinic in Sydney, Australia. Self-report questionnaires measuring worry, distress, depression, anxiety, stress and health-related quality of life (SF-12) were collected. Results Those who had a biopsy recommended were significantly more worried about anal cancer, rated their anal health worse and were less optimistic about their future health than the control group who needed no further medical investigation. The group receiving high grade histology results remained worried about anal cancer at time 3. We found no evidence that general anxiety, depression or quality of life was significantly affected by the process. Conclusions Anal cancer specific worry increases throughout the screening process. Clear communication prior to procedures about the procedure itself, potential adverse events, the recovery process and non-technical explanations of results should be implemented in anal screening programmes.