Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion. Display omitted •Generation of a dominant-negative Rab GTPase library•Identification of Rab regulators of lipoprotein secretion•Rab1b differentially controls the secretion of apolipoproteins and of HCV•ApoE, ApoB100, and HCV may be secreted by distinct transport routes Takacs et al. show that Rab1b, a major regulator of transport from the endoplasmic reticulum to the Golgi, differentially controls the secretion of lipoprotein components ApoE and ApoB100 and infectious hepatitis C virus particles.