Pumpkin seed oil (PSO), a rich source of nutrient contents, is extracted from the seeds of different pumpkin varieties for food and medicine industries. Accordingly, this article was designed to provide an evidence‐based review of the literature, with the purpose of achieving practical conclusions involved the extraction technologies, nutritional properties, and validated biological activities of PSO. From the previous literatures, PSOs contain a large proportion of unsaturated fatty acids, with linoleic acid as the main component, and an amount of tocopherol, phytosterol, and phenolic acids. Some differences in the yield, composition and physicochemical properties of PSO can be associated with the pumpkin's cultivars and the extraction methods. Some novel technologies involved in supercritical fluid extraction, enzyme‐assisted aqueous extraction, ultrasound‐assisted extraction have been gradually replacing the conventional technologies as the most promising method which promote safe, non‐polluting and effective recovery of PSO. This healthy vegetable oil was reported by several in vitro and in vivo studies to have potential protective roles in oxidative stress, inflammation, cancer, and cardiovascular diseases.This article is protected by copyright. All rights reserved.