Background The p24 family is thought to be somehow involved in endoplasmic reticulum (ER)-to-Golgi protein transport. A subset of the p24 proteins (p24α3, -β1, -γ3 and -δ2) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). Methodology/Principal Findings Here we find that transgene expression of p24α3 or p24δ2 specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24α3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24δ2-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. Conclusions/Significance Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.