Background: Infantile colic is period of devastated and inexplicable crying in an apparently healthy infant, leads to drained, bothered and worried parents. It affects 30% of infants worldwide with equivalent frequency in male and female. Aim: To evaluate the impact of an educational program on mothers' knowledge regarding infantile colic syndrome. Methods: Quasi- experimental (pre/posttest) research design used in all of the five maternal and child health care centers. Sample: Included 402 mothers. Two tools; Tool (1): A structure interview questionnaire sheet composed of three parts; part (I): Mothers' personal data; part (II): Infants' data and part (III): Mothers' knowledge about infantile colic syndrome. Tool (II): Infant colic scale, which included total of 22 items in five subscales. Results: It was found that 29.9% and 68.9 of mothers had university education and from rural area. There was improvement of mothers' knowledge in posttest mean score of knowledge =19.46 while it was 8.46 in pretest. There was statistical significance relation between mothers’ total mean score of knowledge and education, occupation and residence in pretest. Conclusion and recommendations: There was improvement in mothers' knowledge after implementation of the educational program. The study recommended availability of educational material in five maternal and child health care centers to increase awareness regarding infantile colic.