We describe incidence and risk factors for pandemic (H1N1) 2009 virus infection in healthcare personnel during the June-September 2009 epidemic in Singapore. Personnel contributed 3 serologic samples during June-October 2009, with seroconversion defined as a ≥4-fold increase in hemagglutination inhibition titers to pandemic (H1N1) 2009. Of 531 participants, 35 showed evidence of seroconversion. Seroconversion rates were highest in nurses (28/290) and lowest in allied health staff (2/116). Significant risk factors on multivariate analysis were being a nurse (adjusted odds ratio [aOR] 4.5, 95% confidence interval [CI] 1.0-19.6) and working in pandemic (H1N1) 2009 isolation wards (aOR 4.5, 95% CI 1.3-15.6). Contact with pandemic (H1N1) 2009-infected colleagues (aOR 2.5, 95% CI 0.9-6.6) and larger household size (aOR 1.2, 95% CI 1.0-1.4) were of borderline significance. Our study suggests that seroconversion was associated with occupational and nonoccupational risk factors.