Wound healing is a physiological process that occurs progressively through overlapping phases. Tissue oxygenation is an important part of the complex regulation for wound healing. Hyperbaric Oxygen (HBO) therapy is a method of increasing oxygen delivery to tissues. The therapy improves tissue oxygenation and stimulates the formation of H2O2 as a secondary messenger for Tumour Necrosis Factor alpha (TNF α), e-NOS, VEGF and Nuclear Factor Kappa Beta phosphorylation (NF-Kb) which play an important role in the rapid transcription of a wide variety of genes in response to extracellular stimuli. This study aims to determine the effects of Hyperbaric Oxygen therapy in enhancing the expressions of e-NOS, TNF-α, VEGF and wound healing. This study is an animal study with a'randomized control group of pre-test and post test design'on 28 Wistar rats. Randomly, the rats were divided into 4 groups with 7 rats in each group. The HBO treatment group 1 received 5 sessions of HBO 2.4 ATA in 3× 30 minutes; the HBO treatment group 2 received 10 sessions of HBO 2.4 ATA in 3× 30 minutes; and each of the control groups were without HBO. Each of the 28 male rats were given a full thickness excisional wound of 1× 1cm. Examinations of e-NOS, TNF-α, VEGF expressions and wound healing were performed on day-0 (pre-HBO) and day-5 HBO or on day-0 (pre-HBO) and day-10 HBO. The resultsshowthat the Hyperbaric Oxygen therapy can improve e-NOS (p= 0.02), TNF-α (p= 0.02), VEGF expression (p= 0.02) and wound healing (p= 0.002) significantly in the provision of HBO 2.4 ATA for 3× 30 minutes in 5 sessions over 5 consecutive days. While the 10 sessions of HBO 2.4 ATA for 3× 30 minutes over 10 consecutive days only increase e-NOS (p= 0.02), TNF-α (p= 0.04), VEGF expression significantly (p= 0.03) but do not improve wound healing significantly (p= 0.3) compared with no HBO. The study concludes that HBO can improve the expressions of e-NOS, TNF-α, VEGF and wound healing in the provision of HBO 2.4 ATA for 3× 30 minutes in 5 sessions, while the 10 sessions of HBO 2.4 ATA for 3× 30 minutes only increase e-NOS, TNF-α, VEGF expression but do not improve wound healing.