Although systematic colorectal cancer screening is efficacious, many programmes suffer from low uptake. Few behavioural or attitudinal factors have been identified as being associated with participation in colorectal cancer screening. We explored knowledge, beliefs about cancer, subjective health literacy, emotional attitudes to screening, and social influences among individuals invited to a population-based screening programme. Regression modelling of a cross-sectional survey of 2299 individuals (users and non-users) of a population-based Faecal Immunochemical Test (FIT) screening programme in Dublin was conducted. Questions were derived from previous theoretically-informed qualitative work and assessed using previously used and validated measures. The primary outcome variable was uptake status (User/Participation or Non-User/Non-participation); multivariable logistic regression was used to estimate the odds ratios (OR) for screening participation. Stronger fatalistic beliefs independently predicted lower uptake (OR = 0.94; 95% CI 0.90–0.98; P = 0.003). Those aged <65 who disagreed that “cancer can often be cured” also had lower uptake (OR = 0.43; 95% CI 0.22–0.82: P = 0.017). Agreement that the test was disgusting and tempting fate predicted lower uptake (OR = 0.16: 95% CI 0.10–0.27: p < 0.001), while the influence of a partner on decision to be screened was associated with higher uptake (OR = 1.32; 95% CI 1.15–1.50: P < 0.001). Negative cancer-related and screening-related beliefs and emotions are associated with non-participation in FIT (−based screening). Research is warranted to explore if these negative beliefs and emotions are modifiable and, if so, whether this would improve screening uptake. The association between the influence of a partner and screening participation present a challenge around improving uptake among those not in co-habiting relationships.