Vitamin D deficiency is a worldwide problem that is largely unknown by majority of population and its prevalence is high even in developed and sunny countries. The purpose of the study was to assess mothers' awareness regarding vitamin D deficiency among their infants in Kalyobia Governorate. Design: Descriptive correlational research design was utilized in this study. Setting: This study was conducted at 4 Health Care Centers in Kalyobia Governorate :Benha 1, Toukh, Bahtem 1 and Begam Health Care Center. Subject: Purposive sample was used in this study; the total sample included 362 mothers. Instruments of data collection: I): An interviewing questionnaire which consists of three parts to assess a): Social characteristics b): Knowledge about vitamin D and its deficiency and, c): Practices regarding prevention of vitamin D deficiency, II): Scale to measure attitude about importance of vitamin D and its supplementation. Results: Regarding mothers' knowledge, 56.9% of the mothers had poor knowledge, 76.8% of mothers had unsatisfactory practices regarding prevention of vitamin D deficiency, and 64.4% of the mothers had positive attitude regarding importance of vitamin D and its supplementation. Conclusion: There was a highly statistically significant relation between total knowledge score of mothers and their age, educational level, occupation, monthly income, and residence. There was a positive statistically significant correlation between total knowledge score, total attitude score and total practice score about vitamin D deficiency. Recommendations: All health care professionals are encouraged to educate mothers the importance of vitamin D and the consequences of its deficiency through health educational program during postnatal or well-baby visits to assure healthy population.