Background: A persistent, crippling disease known as oral submucous fibrosis (OSMF) is distinguished by fibrosis of the juxta-epithelium. It has been proposed that betel quid chewing and its malignant transformation of OSMF are associated with hypoxia, a major micro-environmental component in OSMF. The primary regulator of hypoxia in cellular response, HIF-1α is significantly up-regulated in several fibrotic disorders, including OSMF. Objective: The objective of this study was to examine the expression of the protein HIF-1α in the salivary samples of healthy controls and stage III OSMF. Methods: It was a case-control study carried out in the Department of Oral Medicine, Ziauddin University, Karachi from July 2020 to May 2021. There were 57 participants in the present study, 29 healthy controls and 28 patients with stage III OSMF. The method of non-probability consecutive sampling was employed. Demographic information was gathered using a standardized baseline questionnaire. The enzyme-linked immunosorbent assay was used to measure the amount of HIF-1α present in saliva. To find out if the data was distributed normally, the Shapiro-Wilk test was performed. To compare the expression of HIF-1α protein between the two study groups, the Mann-Whitney test was used. Results: In the stage III OSMF group, the mean salivary HIF-1α value was 25.64±1.27 ng/ml, while in the control group, it was 12.30 ± 1.58 ng/ml. The Mann-Whitney U-test revealed a statistically significant difference (p< 0.001). A significant negative correlation (r = -0.526, p = 0.000) was seen in the individual group analysis between salivary HIF-1α and mouth opening. Conclusion: In comparison to the healthy individuals, the stage III OSMF group had higher levels of HIF-1α protein. The results of our investigation suggest that HIF-1α could be a valuable marker of malignant transformation in OSMF.