The antihistaminic, antiserotonergic drug cyproheptadine (CPH) is known to inhibit insulin synthesis in vivo and in vitro. This inhibition of insulin synthesis occurs without a commensurate decrease in preproinsulin mRNA (PPImRNA) levels, suggesting a post-transcriptional mechanism of action. The goal of the present study was to investigate the direct effects of CPH on translation of PPImRNA in RINm5F cells. Results produced using a subcellular fractionation technique followed by real-time RT-PCR indicated that a 2-h 10 μM CPH treatment resulted in a decrease in the percentage of cellular PPImRNA associated with endoplasmic reticulum (ER) bound polysomes and increases in the percentages of translationally uninitiated and monoribosome-associated PPImRNA. These alterations in PPImRNA distribution were found to be concentration-dependent, chemical structure-specific, and reversible with a time course consistent with a previously reported CPH-induced inhibition of insulin synthesis. Further investigations to examine the possible effect of CPH on translation initiation were then undertaken by examining the phosphorylation state of the translation initiation factors eIF2α, eIF4E, and 4E-BP1 after CPH treatment. CPH (10 μM) treatment resulted in increased phosphorylation of eIF2α, and decreased phosphorylation of both eIF4E and 4E-BP1. These changes are all consistent with decreased initiation of translation. Taken together, these results suggest that the inhibition of insulin synthesis known to be elicited by CPH treatment of RINm5F cells and intact animals involves alterations of initiation factor phosphorylation leading to a decrease in insulin synthesis and of stored insulin in insulin-producing cells.