BACKGROUND: Although the routine testing of pregnant women for the Human Immunodeficiency Virus (HIV) antibody is an integral part of Mother to Child Transmission (MTCT) prevention programs, there is still a high rate of MTCT that can be attributed to early acute infection or existence of infected HIV antibody sero- negative pregnant women. AIM: The current study assessed the prevalence of HIV P24 antigens and MTCT rate among pregnant women with HIV antibody sero-negative status aged between 20 years to 49 years in Calabar, Nigeria. METHODOLOGY: About 5ml of blood samples were collected from consented 400 apparently healthy pregnant women and initially analyzed for HIV 1 & 2 antibodies using DetermineTM HIV-1/2, Stat-Pak HIV-1/2 and HIV UniGold rapid test kit respectively and P24 antigen using DetermineTM HIV-1 & 2 P24 Ag/ Ab Combo test kit. RESULTS: About 12 (3%), 10 (2.5%) and 9 (2.25%) subjects were reactive to Determine HIV 1&, Stat Pak and UniGold test kits respectively with no statistically significant difference between results among study groups (p=0.3065).While 15 (3.75%), 12 (3.0%) and 10 (2.5%) respectively of the 388, 390 and 391 subjects who were initially non-reactive for the three HIV antibody kits became reactive to HIV P24 core antigens with no statistically significant difference between the three antibody test kits and HIV P24 antigen tests (P=0.901). CONCLUSION: The current study found that the prevalence rate of HIV 1&2 P24 core antigen was 1.25 % and MTCT rate was 7.75% in the study population.