Viruses are obligate, intracellular pathogens that hijack host cell machinery to replicate. Innate immunity is the first line of defense against such perceived threats through recognition of broadly conserved pathogen signatures. Tetherin (also known as BST2/CD317/HM1.24) is an innate immune factor that senses and restricts egress, the final step of viral replication. Tetherin potently reduces cell-free virus spread by indiscriminately “tethering” particles at the cell surface via direct anchoring to the host membrane. The majority of previous studies on Tetherin focused on elucidating the minimal structural features necessary for tethering viral particles and understanding how viruses counter Tetherin function. While the cytoplasmic tail of Tetherin is dispensable for restricting virus release in the absence of certain viral antagonists, our work, in part, has focused on conserved residues within the tail, hypothesizing these conserved features could provide insight into previously under-studied biology, including a poorly characterized signaling function. In Chapter 2 we show that human Tetherin can exist as two alternatively translated isoforms [long (l-) and short (s-)]. s-Tetherin lacks the first 12 amino acids, a difference that confers functional differences. We found s-Tetherin to be exquisitely resistant to downregulation by the best characterized Tetherin antagonist, HIV-1 Vpu. l-Tetherin, which is sensitive to Vpu, was shown to be a potent activator of NF-κB. A dual-tyrosine motif, unique to l-Tetherin, was identified as an important determinant. In Chapter 3 we describe additional determinants of signaling. Interestingly, we identified compensatory mutations that produce a tyrosine-independent signaling-competent Tetherin called SY. We demonstrate that SY Tetherin utilizes a similar signaling pathway as wt Tetherin. We believe this mutant, at the very least, can be used as a tool to further elucidate how Tetherin engages signaling machinery. Moreover, the identified compensatory changes in the cytoplasmic tail may be useful in exploring what we hypothesize to be a previously uncharacterized regulatory motif. In this dissertation, we explored two novel aspects of Tetherin biology. Our understanding of how Tetherin isoform expression and Tetherin-mediated signaling is regulated could provide mechanisms by which we can target and enhance immune responses during infection by clinically relevant viruses.