Shift labor, with hours outside of the regular diurnal work cycle, is used to facilitate round-the-clock emergency healthcare services. This impacts the circadian rhythm, which can potentially activate the pro-inflammatory responses of the immune system. This study aimed to find out the levels of pro-inflammatory markers in night shift healthcare workers and find risk factors associated with their increase. 303 participants (doctors, nurses, technicians, health care aids) were recruited with 169 participants nightshift and 134 dayshift workers. Pro-inflammatory markers Highly Sensitive C-Reactive Protein (hsCRP) and Total Leukocyte Count (TLC)] were estimated. Factors were compared by t test, Wilcoxon Rank Sum test and Chi square test. Risk factors for hsCRP elevation were identified by logistic regression analysis. Pro-inflammatory markers like hsCRP and TLC were significantly increased in night shift health workers in comparison to the day shift workers (hsCRP 0.30± 0.33 mg/dl vs 0.10± 0.09 mg/dl, TLC 8181.06± 1181.53 /cu mm vs 7473.80± 1018.81 /cu mm) respectively. Multivariate logistic regression analysis showed night shift work (OR 48.20 95% CI (4.99-465.61), age (OR 1.13 95% CI (1.01-1.26)) and BMI (OR 1.18 95% CI (1.06-1.32)) as independent predictors of elevated hsCRP after adjusting for other risk factors. Hence, night-shift work is an independent risk factor for a rise in pro-inflammatory markers. These findings might aid health care workers and policymakers strategize methods to tackle the challenges through providing support programs, counselling sessions to avoid ailments and deliver health services in a better way.