Durum wheat is a source of both macronutrients and micronutrients, including starch, proteins, fiber, vitamins, and minerals. It is also an important source of carotenoids. Its high carotenoid content makes it ideal for pasta production. Carotenoids are known for their health benefits, which include antioxidative and anti-inflammatory properties. To exert any of these health benefits, potentially absorbed dietary carotenoids must first be bioaccessible. Three Canadian durum wheat varieties (AAC Spitfire, CDC Precision, and Transcend) milled to semolina (RS) and whole flour (WWF) were processed into pasta and cooked to Al dente (Al), fully cooked (FCT), or overcooked (OC) to assess the influence of the food matrix and food processing on carotenoid bioaccessibility and absorption using a static in vitro digestion model. Carotenoid retention after cooking, digestive stability, uptake efficiency, and antioxidant activity were also evaluated using the human Caco-2 cell model. Carotenoid composition of the three wheat varieties was examined using HPLC. The wheat varieties differed in their initial carotenoid contents, with lutein being the predominant carotenoid compound. On average, carotenoid contents decreased by 12-31% after pasta extrusion, and further decreases were observed after cooking. Upon digestion, bioaccessibility of lutein, ranged from 124.5-156.9% in RS and 104.0 -139.1% in WWF pasta. Carotenoids in pasta cooked to Al were significantly (P<0.05) more bioaccessible than FCT and OC, although OC pasta promoted higher extractability. Higher cooking stability of lutein was found in the Transcend variety (75.6%) compared to Spitfire (74.2%) and Precision (69.6%), resulting in its significantly high (P<0.05) lutein bioaccessibility, highlighting the influence of the food matrix. Lutein cellular uptake was low, ranging from 3.4-7.9% for RS pasta and 3.7-5.2% for WWF samples. The cellular antioxidant activity of digested and undigested carotenoids demonstrated their efficiency in attenuating reactive oxygen species-induced oxidative stress, especially at low concentrations. Our results showed that the magnitude of carotenoid bioaccessibility depended on the flour type, the carotenoid physical properties, the cooking duration, and the wheat variety. Knowledge gained from this research provides a foundation for further research into the factors that influence carotenoid bioaccessibility in cereal grain food products.