Changes in cellular and synaptic plasticity related to learning and memory are accompanied by both upregulation and downregulation of the expression levels of proteins. Both de novo protein synthesis and post-translational modification of existing proteins have been proposed to support the induction and maintenance of memory underlying learning. However, little is known regarding the identity of proteins regulated by learning that are associated with the early stages supporting the formation of memory over time. In this study we have examined changes in protein abundance at two different times following one-trial in vitro conditioning of Hermissenda using two-dimensional difference gel electrophoresis (2D-DIGE), quantification of differences in protein abundance between conditioned and unpaired controls, and protein identification with tandem mass spectrometry. Significant regulation of protein abundance following one-trial in vitro conditioning was detected 30 min and 3 h post-conditioning. Proteins were identified that exhibited statistically significant increased or decreased abundance at both 30 min and 3 h post-conditioning. Proteins were also identified that exhibited a significant increase in abundance only at 30 min, or only at 3 h post-conditioning. A few proteins were identified that expressed a significant decrease in abundance detected at both 30 min and 3 h post-conditioning, or a significant decrease in abundance only at 3 h post-conditioning. The proteomic analysis indicates that proteins involved in diverse cellular functions such as translational regulation, cell signaling, cytoskeletal regulation, metabolic activity, and protein degradation contribute to the formation of memory produced by one-trial in vitro conditioning. These findings support the view that changes in protein abundance over time following one-trial in vitro conditioning involve dynamic and complex interactions of the proteome.