© 2015 IMSS. Background and Aims: In April 2009, a new strain of influenza A(H1N1) was identified in Mexico and in the U.S. In June 2009, WHO declared this a pandemic. Health care workers constituted a risk group for their close contact with infected individuals. The aim was to estimate seropositivity for A(H1N1)pdm09 in health staff at the Instituto Mexicano del Seguro Social. Methods: A two-stage cross-sectional study, before and after vaccination in the same workers, was performed on a random sample of health-care workers. A socio-occupational questionnaire was applied and serum antibodies against influenza A(H1N1)pdm09 were determined through neutralization of retroviral pseudotypes; two logistic regression models for both were constructed. Results: The average (median/mean) age of 1378 participants from 13 work centers was 41.7 years and 68.7% (947) were women. Seroprevalence for the first stage was 26.5% (365) (7.4-43%) vs. 20.8% (11) in a control group from the blood bank; for the second stage, the vaccinated group was 33% (215) (18.2-47%) and 27% (196) (11.6-50%) for the unvaccinated group. In regression models, seropositivity was associated with occupational exposure to suspected influenza infected patients, being physicians, and being vaccinated. Conclusions: Seropositivity against pandemic virus is similar to what was reported, both for vaccinated (2.8-40.9%) and unvaccinated (18.8-64.7%). Low seroprevalence in the vaccinated group indicates that between 67% and 73% were susceptible to infection. Given the relatively low vaccine-induced seropositivity, it is imperative to increase, hygiene and safety for health staff and at-risk populations, and strengthen epidemiological surveillance.