Benign prostatic hyperplasia (BPH) commonly occurs in middle age to older sexually intact male dogs. Recently, alternative medical treatment of canine BPH using intraprostatic injection of botulinum toxin type A has been reported. However, in terms of animal welfare, it remains questionable when applied to clinical use. This study aimed at evaluating short-and long-term effect of dogs receiving intraprostatic injection of botulinum toxin type A (BOTOX®). Six intact dogs with no clinical signs of BPH were received 100 units of botulinum toxin type A by transabdominal intraprostatic injection using ultrasound guidance (50 units of each prostatic lobe). Pain scoring, evaluation of white blood cell counts, cortisol concentrations, observation of urination behavior before (24 and 12 hours) and at 2, 4, 6, 12, 24, 48 and 72 hours post-injection were recorded. Short-term effect of botulinum toxin type A on dimension of prostate were measured by ultrasound and MRI at 24 hours before injection and 3 after injection. Long-term effect of botulinum toxin type A on dimension of prostate were measured by ultrasound (at 30, 60, 90 and 120 days). No significant differences in pain score, inflammation condition, stress conditions and urination behavior between before and after injection were found. These findings suggested that, during 3 days post-injection, injection of botulinum toxin type A intraprostatically caused no acute discomfort/pain to animals. No significant change of prostate volume in long-term effect was found.