Abstract: Objective: To investigate the effects of individualized high-definition transcranial direct current stimulation (HD-tDCS) on brain function in individuals with chronic ankle instability (CAI). Methods: Thirty-four CAI were recruited and randomly divided into either real stimulation group or sham stimulation group (n=17 in each group), the individualized HD-tDCS stimulation (20 minutes of individualized current intensity stimulation) and sham stimulation (only 60 seconds of fade-in and fade-out at the beginning of stimulation) were conducted in these two groups, respectively. The participants’ functional magnetic resonance imaging (fMRI) data were collected before and after stimulation. Two-way repeated measures ANOVA was used to examine the effects of individualized HD-tDCS on brain function in individuals with CAI. Results: All participants completed the assessments and no unexpected side effects or adverse events were reported. The blinding effect was good as the total error rate of subjective guesses in stimulation was 44.12%. Compared with pre-intervention and sham stimulation: 1) Single individualized HD-tDCS enhanced the cortical activation of bilateral inferior parietal lobules, bilateral posterior cingulate gyri, bilateral precentral gyrus, and bilateral supplementary motor area (SMA) during the dorsi-plantarflexion task (SMA); 2) single individualized HD-tDCS significantly improved the functional connectivity between the right SMA and the left cerebellum, the left medial frontal gyrus and the right cingulate gyrus, and enhanced the functional connectivity between the left precentral gyrus and the left cerebellum. Conclusions: This study established an HD-tDCS protocol for treating chronic ankle instability, and the results showed that the individualized HD-tDCS can significantly improve brain function (improvement of cortical activation and functional connectivity) in CAI, suggesting that this protocol has certain effects in restoring brain function in CAI.