Osteoarthritis is characterized by persistent pain and dysfunction in the joints due to joint deformity and degradation. This study intended to develop a new microemulsion formulation of hyaluronic acid incorporating cetyl myristoleate for intra-articular administration and pain control related to arthritis. A titration method was utilized to formulate the microemulsions. The formulations were assessed for clarity, pH, droplet size, polydispersity index, zeta potential, and in vitro release analysis. Additionally, CHON-001 healthy human cartilage fibroblast cells were assessed for cytocompatibility. The chosen formulation was examined in vivo in rats. The characterisation investigations revealed that the droplet diameters of the blank, hyaluronic acid loaded and both hyaluronic acid and cetyl myristoleate added as an excepient to the formulation used in in vivo studies were found 25.23 ± 3.14 nm, 36.35 ± 2.10 nm and 78.8 ± 0.4 nm, respectively. PDI values of the mentioned formulations were 0.298 ± 0.035, 0.232 ± 0.006 and 0.250 ± 0.032. The drug concentration in the formulations varied from 9.7 ± 0.2 to 9.9 ± 0.2 mg/mL. Cell culture tests demonstrated that the formulations had cytocompatible profiles. In vivo studies, Mankin scoring was performed to both show the occurrence of osteoarthritis and to demonstrate improvement. At the end of the study, it was observed that the hyaluronic acid-loaded cetyl myristoleate added formulation statistically significantly increased the improvement compared to the control group and the market product. MMP3 and caspase-3 positive cells were examined by immunohistochemical method. In the results obtained, the addition of cetyl myristoleate as an excipient to the formulation loaded with hyaluronic acid showed a synergistic effect and decreased MMP3 expression in a statistically significant way. However, the number of Caspase-3 positive cells was also significantly reduced in the group to which the formulation was applied. Transmission electron microscope images revealed that in the osteoarthritis animal group, cell integrity were disrupted, microvilli structures degenerated and vacuolization was detected in the cytoplasm of the chondrocytes. The cells showed normal morphology when compared with the patient in the animal groups.In conclusion, the created microemulsion technology may provide a more effective and prolonged therapy of hyaluronic acid in the treatment of osteoarthritis compared to conventional medications.