In recent years 3D-bioprinting technology has been developed as an alternative to animal testing. It possesses a great potential for in vitro testing as it aims to mimic human organs and physiology. In the present study, an alginate-gelatin-Matrigel based hydrogel was used to prepare 3D-bioprinted HepaRG cultures using a pneumatic extrusion printer. These 3D models were tested for viability and metabolic functions. Using 3D-bioprinted HepaRG cultures, we tested the toxicity of aflatoxin B1 (10 or 20 μM) in vitro and compared the results with 2D HepaRG cultures. There was a dose-dependent toxicity effect on cell viability, reduction of metabolic activity and albumin production. We found that 3D-bioprinted HepaRG cultures are more resistant to aflatoxin B1 treatment than 2D cultures. Although the metabolic activities were reduced upon treatment with aflatoxin B1, the 3D models were still viable and survived longer, up to 3 weeks, than the 2D culture, as visualized by fluorescence microscopy. Furthermore, albumin production recovered slightly in 3D models after one and two weeks of treatment. Taken together, we consider using 3D-bioprinting technology to generate 3D tissue models as an alternative way to study toxicity in vitro and this could also provide a suitable alternative for chronic hepatotoxicity studies in vitro.