Lamins form the nuclear lamina, which is important for nuclear structure and activity. Although posttranslational modifications, in particular serine phosphorylation, have been shown to be important for structural properties and functions of lamins, little is known about the role of tyrosine phosphorylation in this regard. In this study, we found that the constitutively active Src Y527F mutant caused the disassembly of lamin A/C. We demonstrate that Src directly phosphorylates lamin A mainly at Tyr45 both in vitro and in intact cells. The phosphomimetic Y45D mutant was diffusively distributed in the nucleoplasm and failed to assemble into the nuclear lamina. Depletion of lamin A/C in HeLa cells induced nuclear dysmorphia and genomic instability as well as increased nuclear plasticity for cell migration, all of which were partially restored by re-expression of lamin A, but further promoted by the Y45D mutant. Together, our results reveal a novel mechanism for regulating the assembly of nuclear lamina through Src and suggest that aberrant phosphorylation of lamin A by Src may contribute to nuclear dysmorphia, genomic instability, and nuclear plasticity.