Telomere shortening occurs with aging in immune cells and may be related to immunosenescence. Exercise can upregulate telomerase activity and attenuate telomere shortening in immune cells, but it is unknown if exercise impacts other immune tissues such as the thymus. This study aimed to examine human telomerase reverse transcriptase (hTERT) alternative splicing (AS) in response to aging and exercise in thymus tissue. Transgenic mice with a human TERT bacterial artificial chromosome integrated into its genome (hTERT-BAC) were utilized in two different exercise models. Mice of different ages were assigned to an exercise cage (running wheel) or not for 3 weeks prior to thymus tissue excision. Middle-aged mice (16 months) were exposed or not to treadmill running (30 min at 60% maximum speed) prior to thymus collection. hTERT transcript variants were measured by RT-PCR. hTERT transcripts decreased with aging (r = - 0.7511, p  0.05). In summary, thymic hTERT expression is reduced with aging. Exercise counteracted a shift in hTERT AS ratio with age. Our data demonstrate that aging impacts telomerase expression and that exercise impacts dysregulated splicing that occurs with aging.