The HIV-1 transactivator of transcription (TAT) protein enhances beta amyloid (Aβ42) neurotoxicity and may accelerate Alzheimer’s disease (AD)-related neuronal damage, yet its impact on nuclear architecture remains unclear. In this study, we examined the mechanism by which TAT–Aβ42 affects nuclear integrity. Exposure to TAT–Aβ42-induced a marked elevation in intracellular calcium levels, which subsequently activated cathepsin L (CL), a lysosomal cysteine protease. Activated CL cleaved nuclear lamins, leading to nuclear envelope disruption and altered nuclear morphology. Both calcium chelation and pharmacological inhibition of CL significantly reduced lamin cleavage, highlighting a calcium-dependent CL-mediated pathway. These findings identify a novel mechanism by which TAT–Aβ42 compromises nuclear architecture, providing mechanistic insight into how HIV infection may exacerbate neurodegenerative processes in AD.