Objectives: The current study aimed to investigate and evaluate the knowledge, attitude, awareness, and motivation about blood donation among academic staff in health colleges in Southern region of Saudi Arabia. Methods: A cross-sectional descriptive study was conducted in two universities in the Southern region of Saudi Arabia. The participants were the academic staff in health colleges at King Khalid University, Abha, and Jazan University, Jazan, during 2016. A validated, well-structured questionnaire was used to collectdata. Bivariate logistic regression analysis and odds ratio (OR) were used to assess the knowledge, attitude, awareness, and motivation about blood donation among the study participants. Results: The results showed vast knowledge about blood groups among academic staff, which was significantly associated with their educational level (OR, 4.161; 95% confidence interval [CI], 2.313– 7.484). Knowledge of the Rhesus blood group system is highly significantly associated with age and educational level of academic staff (OR, 0.018; 95% CI, 0.002–0.142 and OR, 0.311;95% CI, 0.150– 0.643). Awareness of the minimum requirements of blood donation is significantly associated with educational level and profession among academic staff (OR, 1.919;95% CI, 1.482–2.485 and OR, 1.258;95% CI, 1.059–1.494). Conclusion: This study provides a deep insight into the blood donation process among academic staff members in health colleges. The outcome of the study currentreflects an extensive knowledge and positive attitude toward blood donation. The academic staff could play a key role in increasing the awareness about blood donation among their students, which could possibly reflect on the society.