Seventy-two serum samples were collected from 14 healthy African elephants (Loxodonta africana), including three calves, to test for 25-hydroxyvitamin D [25(OH)D] as well as for performing biochemical panels. Samples were collected between July 1997 and January 2008 to establish normal 25(OH)D values for the species and to examine the relationship of season and time on these values. Although the number of samples from the calves was small (n  =  7), there was no statistically significant difference in the mean 25(OH)D levels between adults and calves (15.7 ± 7.7 ng/ml versus 17.1 ± 5.8 ng/ml, P > 0.05, respectively). The comparison of mean and individual values among seasons showed some variation, but was not statistically different; therefore, all values were combined for further analyses. The mean value of 25(OH)D for all samples was 15.8 ± 7.5 ng/ml (n  =  72), with a 95% confidence interval of 14.0–17.6 ng/ml. There did not appear to be a direct correlation between 25(OH)D levels and calcium (Ca), phosphorus (P), or calcium∶phosphorus ratio (Ca∶P) based on regression analyses (P < 0.05). Values measured approximated normal distributions. Mean calcium value was 10.5 ± 0.6mg/dl (n  =  61); mean phosphorus value was 5.2 ± 0.8 mg/dl (n  =  50); and mean Ca∶P was 2.06 ± 0.34. Since all animals appeared healthy during the course of sample collection, and bone density on foot radiographs was assessed as good, the results are considered to be normal for this herd. With the incidence of joint disease in older elephants, and metabolic bone disease in hand-reared calves, these values will provide a basis for further studies of calcium metabolism in elephants.