Tropomyosin (Tpm) 1 and 2 are important in the epithelial mesenchymal transition of lens epithelial cells; however, the effect of Tpm1 depletion during aging remains obscure. We analyzed the age-related changes in the crystalline lens of Tpm1- conditional knockout mice (Tpm1-CKO). Floxed alleles of Tpm1 were conditionally deleted in the lens, using Pax6-cre transgenic mice. Lenses of embryonic day (ED) 14, postnatal 1-, 11-, and 48-week-old Tpm1-CKO and wild type mice were dissected to prepare paraffin sections, which subsequently underwent histological and immunohistochemical analysis. Tpm1 and α smooth muscle actin (αSMA) mRNA expression were assessed using RT-PCR. The homozygous Tpm1-CKO (Tpm1) lenses displayed a dramatic reduction in Tpm1 transcript, with no change to αSMA mRNA expression. Tpm1 mice had small lenses with disorganized, vesiculated fiber cells, and loss of epithelial cells. The lenses of Tpm1 mice had abnormal and disordered lens fiber cells with cortical and peri-nuclear liquefaction. Expression of filamentous-actin was reduced in the equator region of lenses derived from ED14, 1-, 11-, and 48-week-old Tpm1 mice. Therefore, Tpm1 plays an integral role in mediating the integrity and fate of lens fiber differentiation and lens homeostasis during aging. Age-related Tpm1 dysregulation or deficiency may induce cataract formation.