Dietary oils are important sources of macronutrients and micronutrients, which can affect health. Sacha inchi oil (SIO), a type of edible oil extracted from Plukenetia volubilis, a plant native to tropical regions, is reported to be rich in omega-3 fatty acids. Previous studies have shown that omega-3 fatty acids are essential, offering numerous health benefits, including improvements in metabolic syndrome, cardiovascular disease, and gut microbiota. Typically, omega-3 sources are animal-based, especially fish oil and cod liver oil. SIO presents an interesting plant-based source of essential fatty acids. However, the compounds and effects of SIO on health and safety are not yet clear. In this study, the effects of SIO were studied both in vitro and in vivo. In vitro, we investigated the chemical compounds by evaluating the fatty acid and antioxidant content. In the in vivo study, the oil supplements were administered orally to male Sprague-Dawley rats continuously for 12 weeks. The effects were also evaluated through measurements of the body composition, fatty acid profile, liver enzymes, and morphology and lipid content of organs; the histology of the organs was assessed using H&E and Oil Red O staining. SIO has a high content of omega-3 fatty acids and significant antioxidant capacity. The rats treated with SIO for 12 weeks showed no increased risk of fatty liver or toxicity in any organs. These results indicate that SIO has high levels of alpha-linolenic acid (omega-3) fatty acids and antioxidant compounds, without altering body composition or serum parameters. Furthermore, SIO did not result in any adverse effects in the rats. These results confirm that SIO is a safe and effective source of omega-3, offering benefits for health.