Motion sickness (MS) can have a negative impact on multiple activities including sailing, flying and dancing. Here, a BALB/c mouse model of MS was established to evaluate the therapeutic effects of acclimatization training and microneedle-mediated transdermal delivery of scopolamine on MS. Motion-susceptible mice were selected based on their motion sickness index (MSI) in response to rotational stimulation. Following desensitization, mice were randomly separated into five groups, which were treated with different rotary stimulation for 28 days. The mechanical strength and cytocompatibility of the scopolamine-loaded microneedles were investigated to ensure percutaneous delivery of scopolamine. In addition, the underlying mechanisms of action of MS therapy were examined by measuring the MSI, number of N-Methyl-d-Aspartate Receptor 1 (NMDAR1)-positive cells, concentration of TNF-α and IL-1β in the serum, and c-Fos protein expression levels in the vestibular nuclei. Furthermore, inductively coupled plasma-mass spectrometry (ICP-MS) was employed to determine trace element levels in the serum, liver and kidney. Our results indicated that the combined treatment of acclimatization training and scopolamine-loaded microneedles could effectively alleviate the symptoms of MS by inhibiting the expression of c-Fos protein and inflammatory factors, reducing NMDAR1 expression levels in the vestibular nuclei, and stabilizing trace element levels in the liver, kidney and serum.