Background: There are few studies regarding the time-dependent effectiveness of topical calcium gluconate treatment for the experimental hydrofluoric acid (HF) burn. Methods: For producing a HF-burn, a drop of 46% or 23% HF solution was put on the back of rats for 3 minutes followed by washing with tap water. Calcium gluconate jelly or ointment was then applied to the burn area at various time intervals. The ointment was applied once a day thereafter. Results: The topical calcium gluconate treatment was much more effective if the application was started within 3 hours. On the other hand, if the application was started after 6 hours or more, there was no difference between the groups and the non-treatment groups. Conclusion: It is suggested that practioners should be ready to prepare quickly the calcium gluconate ointment to treat a HF burn, since the calcium gluconate ointment is not commercially available in Japan.