Significance Glutamate transporters play a crucial role for the recovery of the neurotransmitter glutamate from the synaptic cleft. Thus far, studies of the transport dynamics and detailed information of the working mechanism of this family of transmembrane proteins have been sparse and indirect. Here, we used high-speed atomic force microscopy (HS-AFM) to characterize the transport mechanisms and dynamics of a prokaryotic glutamate transporter homolog Glt _Ph in lipid membranes. We assess transport dynamics as a function of substrate in the imaging buffer and provide direct visual evidence that Glt _Ph transport domains within the trimer are entirely independent.