The study established if 2016 NECO Mathematics multiple choice test items satisfied unidimensionality and local independence assumptions of Item Response Theory; it determined the nature of item characteristics curve in the 2016 NECO mathematics items. It also estimated the item parameter indices of the examination items among Nigerian students. The study further investigated the presence of items that functioned differentially across Igbo, Hausa/Fulani and Yoruba cultural environments and established the presence of test item that functioned differentially for male and female testees. These were with a view to providing information on gender and culture-related differential item functioning in National Examinations Council Mathematics multiple choice questions in 2016. Ex-post facto design was adopted for the study. The population for the study comprised 1,022,474 candidates who enrolled and sat for June/July SSCE 2016 NECO Mathematics Examination. The sample comprised 276,338 candidates who sat for the examination in three purposively selected Geo-political zones in Nigeria (South-West, South-East and North-West). The research instruments used for the study were Optical Marks.