The purpose of this study was to investigate the level of renal function knowledge of primary school pupils in Greece. We conducted a cross-sectional study with a convenience sample of 220 pupils, coming from the 5th and 6th grades of general education schools. A questionnaire consisting of 11 questions was developed from scratch. However, based on an analysis of Cronbach’s alpha values obtained when individual questions were deleted, two questions were removed from the analysis, and only nine remained for analysis and participated in the calculation of the knowledge score. Moreover, the gender and daily habits of pupils regarding water consumption and frequency of urination were recorded. Pupils had a high percentage of correct knowledge about the number of kidneys (95.2%), whether a child may have problems with the kidneys (85.5%) and whether a person can survive with one kidney (68.5%). Low levels of knowledge were observed in the function and role of the kidneys (36.4%), as well as the part of the body where the kidneys are located (30.9%). The median (interquartile range (IQR)) total knowledge score was 6 (5–7), with no difference detected between genders (p = 0.135). A statistically significant difference between pupils of 5th and 6th grades was found but the difference did not seem to be clinically significant (p = 0.035). The present research demonstrates that pupils’ knowledge of renal function and the protection of their kidneys needs improvement.