Local anesthetic injections are an essential tool in dentistry, particularly in pediatric dentistry. The needle penetrating the tissue can cause stress, anxiety, and pain. Studies have shown that using touch may alleviate pain and reduce patient anxiety. Yet, this has not been tested in pediatric dental patients. Therefore, this study examined the effect of hand-holding on children undergoing local anesthetic injections. Its effect on children’s pain perception was tested, with the hypothesis that pain perception would be lower for children whose hand was held by an assistant. Additionally, the study examined whether hand-holding would affect children’s anxiety levels and cooperation. Fifty-five children, who underwent dental treatment within the Department of Pediatric Dentistry at Tel Aviv University, were recruited. The patients were randomly divided into two groups. In the study group, the assistant gently placed her hand on the patient’s hand during the anesthetic injection. In the control group, the same treatment was performed without the hand being placed by the assistant. After the anesthetic injection, the child’s pain and anxiety levels were assessed using visual analog scales (VAS). The patients’ pulse was measured. The level of cooperation was evaluated using the “Frankl” scale. Interestingly, although the trends aligned with this study’s hypotheses, no significant effect of hand-holding on pain, anxiety, or cooperation during anesthetic injections was found.