This investigation focused on how consuming alkaline water affected vitamin D3 toxicity and how it related to rats' motor and neurobehavioral activity as well as some biochemical factors. 15 rats were employed, each allocated into three groups. Tap water was provided to the first group (the negative control), alkaline water to the second group, and alkaline water combined with a weekly intramuscular injection of vitamin D3 in a dose of 100,000 unit/animal  to the third group for a period of 4 weeks. Changes in the neurobehavioural and motor activity of the rats were noted in the open field. The weights of the rats in the vitamin D3 group increased. The number of squares and times they stand on their hind legs, and the number of pocking were all significantly higher in the group of vitamin D3 also vitamin D3 levels increased noticeably and considerably lower levels of total cholesterol, Low density lipoprotein levels were also reduced. This investigation has led us to the conclusion that drinking alkaline water can help people and animals who are taking large amounts of vitamin D3, in addition to its effect in reducing the levels of total cholesterol and Low-density lipoprotein with improving body weight and oxidative stress status.