Chiropractic care is a common treatment modality used in equine practice to manage back pain and stiffness but has limited evidence for treating lameness. The objective of this blinded, controlled clinical trial was to evaluate the effect of chiropractic treatment on chronic lameness and concurrent axial skeleton pain and dysfunction. Two groups of horses with multiple limb lameness (polo) or isolated hind limb lameness (Quarter Horses) were enrolled. Outcome measures included subjective and objective measures of lameness, spinal pain and stiffness, epaxial muscle hypertonicity, and mechanical nociceptive thresholds collected on days 0, 14, and 28. Chiropractic treatment was applied on days 0, 7, 14, and 21. No treatment was applied to control horses. Data was analyzed by a mixed model fit separately for each response variable ( < 0.05) and was examined within each group of horses individually. Significant treatment effects were noted in subjective measures of hind limb and whole-body lameness scores and vertebral stiffness. Limited or inconsistent therapeutic effects were noted in objective lameness scores and other measures of axial skeleton pain and dysfunction. The lack of pathoanatomical diagnoses, multilimb lameness, and lack of validated outcome measures likely had negative impacts on the results.