Background: Musculoskeletal pain is a leading cause of morbidity, low productivity. Thus; not only affecting the individual’s quality of life; it also creates a burden in the health system and affects the productivity of their institution and the country at large. The prevalence of musculoskeletal pain among academic staff in developed countries ranges from 47% to 85%. However, there was a scarce of studies in developing country, particularly in the study area. Purpose: the aim of the research was to assess the burden of musculoskeletal pain and associated factors among Mekelle University academic staff. Patients and Methods: An institutional based cross-sectional study was enrolled on 449 participants with a response rate of 92.2%. Multistage sampling technique was deployed to select representatives. Participants under the selected schools, institutes and departments were selected using random sampling method. Data was collected through face to face interview using structured and standardized Nordic questionnaire by trained data collectors at Mekelle University. Binary logistic regression was used to assess the association between dependent and independent variables. In bivariate logistic regression variables which have P value of <.25 were modeled to multivariate logistic regression. Those variables with P-value of <.05 with 95% CI in multivariate model were taken as statistically significant. Results: This study found that burden of musculoskeletal pain among Mekelle University academic staff in the previous 12 month was 65.2%, and in the last 7 days was 29%. Neck pain (41.5%) was most prevalent followed by low back pain (40.3%). Female gender (OR = 3.02, 95% CI: 1.58-5.76), Body mass index ⩾25 (OR = 3.68, 95% CI: 1.15-11.39), working hours per day (OR = 3.1, 95% CI: 1.54-6.38), and physical inactivity (OR = 3.48, 95% CI: 1.69-7.16), were the independent factors positevly associated with musculoskeletal pain. Conclusion: The burden of musculoskeletal pain among Mekelle University academic staff was common. Female gender, being overweight and obese, working >5 hours per day, and being physically inactive increase the odds of experiencing musculoskeletal pains among academicians. Therefore the university authorities and all academicians are recommended that to take preventable measures of musculoskeletal pain.