Influenza may cause severe complications for pregnant women. In this study antibody response against 2009 H1N1 influenza virus in pregnant women was investigated. This seroprevalance cross sectional and questionnaire based study was conducted using a convenient sampling method. Blood samples of pregnant women were checked for antibodies against 2009 H1N1 influenza virus using hemagglutination inhibition assay. An antibody titer level of ≥ 1:40 dilution was considered as the protective level. 167 (43.60%) of 383 pregnant women who participated in this study had protective antibody levels against this virus. 62 (35.63%) of 3rd trimester, 79 (46.74%) of 2nd trimester, and 21(52.50%) of 1st trimester pregnant women were immune respectively (χ2(for trend) = 8.20, p < 0.004). Lack of protective antibody level was significantly seen more in pregnant women of 3rd trimester of pregnancy (OR = 2.37, CI = 1.09-5.18). Pregnant women with higher education (OR = 1.67, CI = 1.02-2.73) and those with history of anemia (OR = 2.09, CI = 1.18-3.68) had more immunity. Older women (OR = 0.95, CI = 0.91-0.99) and those with history of psychological diseases (OR = 0.19, CI = 0.05-0.70) had less immunity. Vaccination of pregnant women, especially those who are in the higher trimesters of pregnancy, older, or less educated, against the 2009 H1N1 influenza virus should be continued.