Background The world is currently facing a pandemic of vitamin D deficiency especially women in child bearing age, during pregnancy and nursing mothers. One of the major reasons for the worldwide spread of this nutritional disorder has been lack of awareness about the importance of vitamin D and prevention of deficient states across populations. Objectives To assess the knowledge and attitude of mothers attending primary health care center in Cairo towards vitamin D importance, to identify mothers' practices towards vitamin D, its supplementation & sun light exposure. Subjects & Methods A cross sectional study was conducted among mothers attending “100 meters” primary health care center in El-Sherouk city in Cairo. A sample of 195 mothers were interviewed using structured questionnaire to collect data including socio-demographic characteristics, mother’s knowledge about the importance of Vitamin D, mother’s attitude and practices towards taking vitamin D, its supplementation and sun light exposure for herself and her child. Scoring system was designed for knowledge, attitude and practice of mothers. A short targeted health education message was given to the participating mothers about vitamin D importance in the form of flyers. Results Less than one quarter of the mothers (22.6%) had a good knowledge towards vitamin D and its supplementation (total knowledge score percentage > 50%) . The highest knowledge about benefits of vitamin D was “bone health” and the least knowledge recalled by mothers were “prevention of depression and obesity”. Thirty six percent of mothers had a positive attitude towards vitamin D and its supplementation (total attitude score percentage ≥75%) & forty six percent of mothers had a good practice towards vitamin D and its supplementation ( total practice score percentage ≥75%) . More than half of mothers (53.5%) were exposed to the sun from 10 am to 3 pm. The majority of mothers (91.1%) were exposed to the sun more than one hour per week. Only (48.7%) of the mothers gave their children vitamin D supplementation & about half of them (52%) started giving vitamin D supplement to her children since birth. Highly educated mothers had better knowledge and practice score than others (P value < 0.05). Conclusion & Recommendation Less than one quarter of the mothers had a good knowledge towards vitamin D and its supplementation while about three fifths of the mothers had a positive attitude regarding vitamin D and its supplementation & less than half of the mothers had a good practice towards vitamin D and its supplementation. Implementing awareness campaigns and future health education programs may help in building more awareness about the vitamin D importance.