The present study was to evaluate the bioavailability of iron (Fe) fortified in parboiled rice grain, expressed as Fe uptake by Caco-2 cells after in vitro digestion. The bioavailability of Fe-fortified in the rice grain was closely and positively correlated with increasing concentrations of Fe in the grains of the three cultivars (r = 0.96∗∗). The uptakes of the Fe-fortified in parboiled rice milled for 120 s (34.2, 47.7 and 107 ng ferritin mg protein⁻¹ in three cultivars, respectively) were well above those of the unfortified raw (6.1, 4.9 and 5.7 ng ferritin mg protein⁻¹) or parboiled rice (4.7, 3.6 and 4.4 ng ferritin mg protein⁻¹), the high Fe rice line IR68144-2B-3-2-2 (4.0 ng ferritin mg protein⁻¹) and popular Jasmine rice cultivar KDML 105 (3.9 ng ferritin mg protein⁻¹). Increasing milling time and rinsing the Fe-fortified parboiled rice decreased Fe bioavailability, due to their negative effects on total Fe concentrations in the parboiled rice grains, but uptakes were still well above that of their unfortified raw or parboiled rice grains. Rinsing or washing the Fe-fortified and milled rice grains decreased the bioavailability to 85 ng ferritin mg protein⁻¹ in the YRF cultivar, compared to about 100 ng ferritin mg protein⁻¹ in its non-rinsed grains. Dilute acid-extractable (DAE) Fe was linearly, positively correlated with the uptake of Fe assessed by the in vitro digestion/Caco-2 cell technique (r = 0.90∗∗), which can be used as a rapid method for optimizing levels of bioavailable Fe to be fortified in the parboiled rice by parboiled-rice mills if this Fe-fortification technique should be adopted in south and southeast Asia.