Multiple Myeloma (MM) is a malignancy of monoclonal plasma cell proliferation in the bone marrow, which accounts for 10% of all hematologic cancers. This slow proliferating B-cell malignancy accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics, which target rapidly replicating cells. MM remains an incurable disease in need of new therapeutic approaches. We have previously shown that the nucleoside analog, 8-chloro-adenosine (8-Cl-Ado), has significant activity for MM and that its sister compound, 8-amino-adenosine (8-NH2-Ado), appears to be even more potent, exhibiting greater activity in pre-clinical studies. 8-NH2-Ado is cytotoxic to MM cell lines both sensitive and resistant to conventional chemotherapies. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its tri-phosphate form, 8-NH2-ATP, and accumulation of 8-NH2-ATP results in a concomitant loss of endogenous ATP levels. 8-NH2-Ado induces apoptosis in MM cells as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a subG1 DNA content. Here we demonstrate the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. MM cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including ERK1/2, p38 MAPK and Akt kinase, within 30–120 minutes. Cells depleted of ATP by antimycin A or 2-deoxyglucose treatments to mimic 8-NH2-Ado-induced ATP depletion do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may be affecting the activity of protein phosphatases involved in the negative regulation of these signaling molecules. Based on studies using the phosphatase inhibitor okadaic acid, it appears that PP2A may play a role in the 8-NH2-Ado-induced decrease in phosphorylation of p38. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analog drug and appears to be unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical pro-survival and apoptosis-regulating proteins suggest that the modulation of these proteins by de-phosphorylation at early time-points may be an important mechanistic step in 8-NH2-Ado-induced programmed cell death.