Lactose promotes the intestinal absorption of calcium independent of the vitamin D endocrine system. This study investigated the effects of lactose on intestinal alkaline phosphatase (ALP) activity in rats. A total of 66 Sprague-Dawley strain female rats (10 weeks old) were divided into two groups: the control and the lactose groups. Animals in the lactose group were fed the experimental diet, in which the 10% of the diet was replaced with lactose. At 0, 3, 7, 14, and 21 days after beginning the experimental diets, rat intestinal segments from the duodenum, jejunum, and ileum were obtained immediately after sacrifice. The segments were slit open longitudinally, and the mucosa was scraped and used for the enzyme assay. The level of intestinal ALP activity in the jejunum from the lactose group was significantly higher than that from the control group. Two kinds of mRNA of rat intestinal ALP (RTIN-1 and RTIN-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The level of mRNA expression in the jejunum from the lactose group was enhanced, especially of RTIN-2. This result was compatible with the results of enzymatic activity. These findings suggest that lactose affects intestinal Pi metabolism not only directly, but also in an indirect way via regulation of intestinal ALP expression, especially in the jejunum.