Purpose: After medial open wedge high tibial osteotomy (MOWHTO), an increase in the joint space width(JSW) of the medial compartment of the knee joint is commonly observed in clinical practice. Since the JSW is regarded as a surrogate measure for cartilage thickness, it has been suggested that the increment of the medial JSW observed after MOWHTO would have resulted from the cartilage regeneration. However, the association between the change of medial JSW after MOWHTO and cartilage regeneration has not investigated with the second-look arthroscopic examination, which could provide an actual status of the cartilage status. Moreover, the possibility that change in JSW is due to the shift of the weight-bearing axis of the lower limb cannot be excluded. The aim of the present study was to investigate the relevant factors that affect the change of JSW after MOWHTO. It was hypothesized that the change of JSW after MOWHTO would be associated with the change of the weight-bearing condition. Methods: A total of 100 patients who underwent MOWHTO and a following second-look arthroscopic assessment were included in this study. The patients were classified into two groups according to the change of medial JSW between at the time of primary operation(MOWHTO) and at the time of second-look operation: group 1(the patients with increased medial JSW) and group 2 (the patients with maintained or reduced medial JSW). Comparative analysis was performed regarding clinical scores, radiographic parameters, and arthroscopic measurements between the two groups. In addition, for the statistically significant variables, the characteristics of JSW of the knee were further evaluated. Results: There were no differences between the two groups in the preoperative clinical scores as well as the postoperative clinical scores evaluated at the time of second-look operation. Furthermore, there were no significant differences between the two groups regarding the change of cartilage lesion size between the two-time points as well as the cartilage regeneration status. However, the postoperative load-axis deviation and the postoperative medial proximal tibial angle were significantly higher in group 1, suggesting that the increase in medial JSW is possibly related to the postoperative weight-bearing condition. Accordingly, an additional analysis was performed to evaluate whether there were differences in JSW characteristics (medial JSW, lateral JSW, and joint-line convergence angle) according to the position of the postoperative load-axis deviation. The patients were further divided into three groups (group U, under-correction group; group A; acceptable correction group; group O; over-correction group). As a result, there was a statistically significant difference in the postoperative medial JSW and the degree of change in medial JSW, in which the medial JSW increased as the postoperative load-axis deviation located laterally. Medial JSW increased over time in group A and O, whereas it showed a tendency to decrease in group U. On the contrary, there were no differences in lateral JSW and joint-line convergence angle between the groups. Conclusions: The increase in the medial JSW observed after MOWHTO was affected by the postoperative weight-bearing condition, rather than the cartilage regeneration status. Therefore, changes in JSW observed after MOWHTO should not be used as a surrogate measure for cartilage thickness. In addition, for a clinical perspective, it could be recommended to aim the post-operative weight-bearing line to be located at 60-70% of the tibial articular surface from the medial border of the tibia, considering postoperative medial JSW as well as other biomechanical aspects of the knee.