Crystallin proteins serve as both essential structural and protective components of the ocular lens required for its transparency and light refraction. The mouse lens crystallin proteome is represented by alphaA-, alphaB-, betaA1-, betaA2-, betaA3-, betaA4-, betaB1-, betaB2-, betaB3-, gammaA-, gammaB-, gammaC-, gammaD-, gammaE, gammaF-, gammaN-, and gammaS-crystallin proteins encoded by 16 genes. Their mutations are responsible for lens opacification and early onset cataract formation. While many cataract-causing missense and nonsense mutations are known for these proteins, including the human CRYBB3 gene, the mammalian loss-of function model of the Crybb3 gene remains to be established. Herein, we generated the first mouse model via deletion of the Crybb3 promoter that abolished expression of the betaB3-crystallin resulting in disrupted lens morphology with initial phenotypic variability. The lens morphology was evaluated at histological levels and in-depth lens proteomes were analyzed using newborn, 3-week, 6-week, and 3-month-old lenses. These Crybb3-null lens proteomes showed both down- and up-regulation of various cytoplasmic and nuclear proteins with the largest differences found in 3-months lenses. Expression of Smarcc1/Baf155, b, and c proteins were validated by western immunoblotting/immunofluorescence. The betaB3-crystallin promoter region contain multiple binding sites of transcription factors AP-2a, c-Jun, c-Maf, Etv5, and Pax6, and is activated by FGF2 in cell culture experiments. Together, these studies establish the mouse Crybb3 loss-of-function model and its disrupted crystallin and non-crystallin proteomes.