Depression is a common mental illness and a leading cause of disability. The most widely prescribed antidepressant medications are serotonin (5-HT) selective reuptake inhibitors (SSRIs). Although there is much support for 5-HT transporter (SERT) antagonism as a basis of antidepressant efficacy, this evidence is indirect and other targets and mechanisms have been proposed. In order to distinguish SERT-dependent and -independent effects of SSRIs, we developed a knock-in mouse model whereby high-affinity interactions of many antidepressants at SERT have been ablated via knock-in substitution (SERT Met172) without disrupting 5-HT recognition or uptake. Here we utilize the C57BL/6J SERT Met172 model to evaluate SERT dependence for the actions of two widely prescribed SSRIs, fluoxetine and citalopram, in tests sensitive to acute and chronic actions of antidepressants. In the tail suspension and forced swim tests, fluoxetine and citalopram fail to reduce immobility in SERT Met172 mice. In addition, SERT Met172 mice are insensitive to chronic fluoxetine and citalopram administration in the novelty induced hypophagia test (NIH) and fail to exhibit enhanced proliferation or survival of hippocampal stem cells. In both acute and chronic studies, SERT Met172 mice maintained sensitivity to paroxetine, an antidepressant that is unaffected by the Met172 mutation. Together, these studies provide definitive support for an essential role of SERT antagonism in the acute and chronic actions of two commonly used SSRIs in these tests, and reinforce the utility of the SERT Met172 model for isolating SERT/5-HT contributions of drug actions in vivo.