There is growing evidence that 1,25-dihydroxyvitamin D3 is involved in normal brain development. The aim of this study was to examine the impact of prenatal and postnatal hypovitaminosis D on prepulse inhibition (PPI) of acoustic startle in adult rats. We compared six groups of rats: control rats with normal vitamin D throughout life and normal litter size (Litter); control rats with normal vitamin D but with a reduced litter size of two (Control); offspring from reduced litters of vitamin D deplete mothers who were repleted at birth (Birth), repleted at weaning (Weaning) or remained on a deplete diet until 10 weeks of age (Life); or control rats that were placed on a vitamin D-deficient diet from 5 to 10 weeks of age (Adult). All rats were tested in acoustic startle chambers at 5 and 10 weeks of age for acoustic startle responses and for PPI. There were no significant group differences at 5 weeks of age on the acoustic startle response or on PPI. At 10 weeks of age, rats in the Life group only had impaired PPI despite having normal acoustic startle responses. We conclude that combined prenatal and chronic postnatal hypovitaminosis D, but not early life hypovitaminosis D, alters PPI.