PURPOSE: In this study, we investigated whether experimental knee pain alters lower limb kinematics and knee arthrokinematics during gait, and if this motor adaptation depends on the spatial characteristics of the painful stimulus. METHODS: Twenty-one participants walked on a treadmill for 60-s trials, either without stimulation or while experiencing painful electrical stimulation in the medial, lateral or anterior region of the knee. Perceived pain location was analyzed using pain drawing. Gait spatiotemporal parameters, lower limb kinematics, and dispersion of the knee helical axes on the sagittal plane were quantified for each trial and compared between conditions using ANOVAs with repeated measures or Friedman tests. RESULTS: Pain perception was localized in the area the stimulation was applied to. Compared to walking without pain, participants demonstrated reduced knee extension (1.5 ± 1.5 degrees, p = 0.002) and reduced hip extension (0.8 ± 1.1 degrees, p = 0.037) when pain was induced in the anterior region, but not medially or laterally. Anterior knee pain increased the mean distance of the helical axes during late stance (0.7 [0.3, 1.4], p = 0.010), while medial pain increased both mean distance (0.3 [0.1, 0.5], p = 0.037) and mean angle (1.2 ± 1.4, p = 0.010) during early swing. CONCLUSION: Acute, experimental knee pain alters gait kinematics and increases the dispersion of the helical axis. These adaptations depend on the spatial characteristics of the painful stimulus. These adaptations may reflect an attempt of the central nervous system to protect the painful tissue while searching for a less painful movement strategy.