In 3 trials, the effects of dietary supplementation with phytase (PHY) and 25-hydroxycholecalciferol on BW and the blood characteristics of commercial layers that were inoculated prelay (12 wk of age) or at the onset of lay (22 wk of age) with F-strain Mycoplasma gallisepticum were assessed at 34, 50, and 58 wk of age. Experimental layer diets, which included either a basal control diet or the same diet supplemented with 0.025% PHY and 25-hydroxycholecalciferol, were fed from 20 through 58 wk of age. The supplemented diet decreased blood hematocrit values across bird age, inoculation type (sham vs. F-strain M. gallisepticum), and age of inoculation (prelay vs. onset of lay). Phytase- and 25-hydroxycholecalciferol-supplemented diets reduced bird BW in sham-inoculated control birds across bird age and age of inoculation. This effect was not observed in F-strain M. gallisepticum-inoculated birds. Furthermore, across diet (control vs. supplemented) and inoculation type, total plasma protein concentration at 34 wk of age was higher in birds that were inoculated at the onset of lay compared with those inoculated prelay. Diet, inoculation type, and inoculation age had no effect on mortality, reproductive organ histopathological lesion scores, or serum cholesterol and Ca concentrations. In conclusion, throughout lay, the supplementation of commercial layer diets with PHY may lower hematocrit, and inoculation with F-strain M. gallisepticum prelay or at the onset of lay may ameliorate the depressing effects of dietary PHY and 25-hydroxycholecalciferol supplementation on hen BW.