Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.