We earlier reported that coating poorly water-soluble drugs with nano-hydroxyapatite (nano-HAP) improves bioavailability after oral administration. In the present study, we coated BCS Class IV drug acetazolamide (AZ) with nano-HAP (AZ/HAP formulation), and investigated its bioavailability and nano-HAP’s role in promoting it. We tested AZ bioavailability after a single oral dose of the AZ/HAP formulation in rats, followed by a series of in vitro, ex vivo and in vivo testing. The binding state of AZ and nano-HAP was analyzed by gel filtration chromatography. AZ permeability was studied using a Caco-2 cell monolayer assay kit, to test for tight junction penetration, then using an Ussing chamber mounted with intestinal epithelium, both with and without Peyer’s patch tissue, to examine the role of intracellular transport. Fluorescence-labeled nano-HAP particles were administered orally in rats to investigate their localization in the intestinal tract. The area under the blood concentration time-curve in rats was about 4 times higher in the AZ/HAP formulation group than in the untreated AZ group. Gel filtration analysis showed AZ and nano-HAP were not bound. The Caco-2 study showed equivalent AZ permeability for both groups, but without significant change in transepithelial electrical resistance (TEER), indicating that tight junctions were not penetrated. In the Ussing chamber study, no significant difference in AZ permeability between the two groups was observed for epithelium containing Peyer’s patch tissue, but for epithelium without Peyer’s patch tissue, at high concentration, significantly higher permeability in the AZ/HAP formulation group was observed. Fluorescent labeling showed nano-HAP particles were present in both intestinal villi and Peyer’s patch tissue 30 min after oral administration. Our results suggest that nano-HAP’s enhancement of drug permeability from the small intestine occurs not via tight junctions, but intracellularly, via the intestinal villi. Further study to elucidate the mechanism of this permeability enhancement is required.