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Background Cytomegalovirus (CMV) is transmitted by direct contact with body fluids from infected individuals. Transmission of CMV in households, particularly those with young children, contributes significantly to CMV infection in the general population. However, little is known about the contribution of occupational healthcare or childcare exposure to risk of CMV infection. Objectives To determine CMV seroprevalence, incidence of primary infection, and associated risk factors in healthcare and childcare workers. Methods Six electronic databases were searched systematically for publications on CMV infection in healthcare and childcare workers until March 7, 2022. Two authors independently evaluated the literature for quality and inclusion in our analyses. The pooled results for seroprevalence, incidence, and relative risk (RR) were determined using a random effects model. Heterogeneity among studies was quantified and further investigated in subgroup analysis and meta-regression. Publication bias was assessed using funnel plot. Statistical analyses were preformed using R version 4.05. Results Forty-eight articles were included in this meta-analysis (quality assessment: 18 good, 14 fair, and 16 poor). Pooled CMV seroprevalence was 59.3% (95% CI: 49.8–68.6) among childcare workers and 49.5% (95% CI: 40.3–58.7) among healthcare workers, and pooled incidences of primary CMV infection per 100 person-years were respectively 7.4 (95% CI: 3.9–11.8) and 3.1 (95% CI: 1.3–5.6). RR for primary infection compared to controls were 3.4 (95% CI: 1.3–8.8) and 1.3 (95% CI: 0.6–2.7) for healthcare and childcare workers, respectively. The odds of CMV seropositivity were 1.6 (95% CI: 1.2–2.3) times higher for childcare workers compared to controls, but not significantly different between healthcare workers and controls (0.9; 95% CI: 0.6–1.2). CMV seropositivity in both groups was significantly associated with having one or more children residing at home, marital status, ethnicity, and age. Conclusions Childcare workers, but not healthcare workers, have an increased risk of prevalent and incident CMV infection, a risk that is further increased with the presence of at least one child living at home. These findings suggest that enforcing simple, conventional hygienic measures in childcare settings could help reduce transmission of CMV, and that special precautionary measures for preventing CMV infection may not be required for pregnant healthcare workers. Systematic review registration PROSPERO CRD42020139756