Background: Practice nurses in general practice sub-optimally adhere to evidence-based smoking cessation treatment guidelines, but factors explaining their adherence have not yet been investigated. Understanding such factors is important to develop interventions improving practice nurses' smoking cessation guideline adherence and patients' subsequent cessation success. This study explored the association between different socio-cognitive and predisposing factors, and practice nurses' adherence to the Dutch smoking cessation guideline in general (i.e. overall adherence) and to each guideline step individually (i.e. step-based adherence). Methods: A cross-sectional study was conducted among practice nurses (N = 157) in January-March 2015 via web-based questionnaires, assessing constructs from the Integrated Change Model. Spearman's correlations and linear regression analysis were used to identify potential determinants of overall guideline adherence; Mann-Whitney U-tests and logistic regression analyses were used to identify potential determinants of step-based adherence. Results: On average five out of nine steps were completely adhered to by practice nurses; and step-based adherence ranged from 34% to 75%. Overall guideline adherence was associated with high levels of self-efficacy to use a guideline (β = 0.32, P = 0.00), and step-based adherence was additionally associated with spending more time on counselling. Regression results showed positive associations between self-efficacy (8/9 steps) and perceived advantages (7/9 steps) with step-based adherence. Conclusion: This study quantitatively confirmed practice nurses' sub-optimal guideline adherence and found associations between socio-cognitive (self-efficacy and perceived advantages) and predisposing factors (time spent on counselling), and guideline adherence. Detailed insights in these factors offer preliminary directions for intervention development to improve practice nurses' adherence to evidence-based smoking cessation guidelines.