Symptoms of osteoarthritis (OA) afflict approximately 20% of adult dogs in North America. Clinical signs consistent with OA include decreased range of motion of a joint, reduced physical activity, difficulty climbing stairs or onto furniture, and a reduced ability to rise from a lying position. A safe and effective nutraceutical supplement may benefit dogs suffering from OA. Calcium fructoborate (CFB), a mimetic of a naturally occurring molecule, has previously been reported to be safe and effective in humans with joint problems. The objective of this randomized, double-blinded, placebo-controlled study was to evaluate the short-term effects of CFB alone, or in combination with a blend of glucosamine hydrochloride (GH) and chondroitin sulfate (CS), on gait analysis, goniometry, serum inflammatory markers, and owner perception of pain in client-owned dogs. Sixty-four dogs with joint discomfort were recruited and 59 dogs (mean age = 8.42 ± 0.37 yr.; mean BW = 31.11 ± 1.28 kg) completed the study. All procedures were approved by the University of Illinois Institutional Animal Care and Use Committee, and pet owners signed an informed consent prior to study initiation. Dogs were randomly assigned to one of four treatments: placebo (60 mg fructose; n = 15), low dose (69 mg CFB; n = 14), high dose (127 mg CFB; n = 14), or combination (69 mg CFB, 500 mg GH and 200 mg CS; n = 16). Treatments were provided once daily as dietary supplements. Small dogs weighing up to 22.9 kg received 1 capsule/day, while large dogs weighing 23 to 50 kg received 2 capsules/day for 28 days. A physical examination, radiographs, goniometry measurements, gait analysis, blood sample collection, and the canine brief pain inventory (CBPI) questionnaire were performed and administered on days 0 and 28. As expected, a majority (69%) of the dogs were overweight or obese, with a body condition score (BCS) > 6 on a 9-point scale. Dogs fed the low dose (-2.93) and high dose (-2.21) of CFB were shown to improve (P < 0.05) in their ability to rise from a lying position from day 0 to day 28 compared to dogs fed the placebo (0.00), but no difference was observed for dogs fed the combination treatment. Dogs assigned the low dose of CFB also tended to have an improved pain severity score (PSS; -1.46; P = 0.08) and pain at its worst score (-2.14; P = 0.06) from day 0 to day 28 compared to dogs fed the placebo (0.05 and 0.00, respectively). Dogs fed the high dose of CFB had a greater increase (P = 0.05) in serum concentration of soluble receptor for advanced glycation end products (sRAGE) from day 0 to day 28 (7.88 ng/mL) compared to dogs fed the placebo (0.83 ng/mL). All blood metabolites were within reference range except total alkaline phosphatase and corticosteroid-induced alkaline phosphatase, which started and ended at concentrations greater than the upper reference range. Dogs assigned the high dose of CFB tended to have a greater reduction (P = 0.07) in serum chloride from day 0 to day 28 (-1.64 mmol/L) compared to dogs fed the low dose of CFB (0.08 mmol/L). Given the low number of small dogs recruited and the increased variability noted as a result of their inclusion, a sub-analysis of large dogs only was performed. Large dogs fed the low dose were shown to have decreased (P < 0.05) scores for PSS (-1.77) and pain at its worst (-2.45) from day 0 to day 28 compared to the placebo group (0.19 and 0.42, respectively). Large dogs assigned the low dose of CFB tended to have improved scores for pain at its least (-1.27; P = 0.08) and pain on average (-1.82; P = 0.07) from day 0 to day 28 compared to dogs fed the placebo (0.25 and -0.08, respectively), but no difference was observed for dogs fed the high dose or combination groups. Large dogs fed the low dose also were shown to improve (P < 0.05) in their ability to rise from a lying position (-3.09) compared to the placebo treatment (0.25) from day 0 to day 28. Overall, supplementation of CFB alone was well-tolerated and appeared to have potential for joint discomfort mitigation in canines