The Yip1 domain family (YIPF) proteins are homologues of yeast Yip1p and Yif1p, which are proposed to function in ER to Golgi transport. Here, we report the characterization of YIPF3 and YIPF4, homologues of human Yif1p and Yip1p, respectively. Immunofluorescence and immuno-electron microscopy showed that both YIPF3 and YIPF4 are clearly concentrated in the cis-Golgi. While YIPF4 was detected as a single mobility form consistent with its predicted molecular weight, three different mobility forms of YIPF3 were detected by western blotting. Biochemical and immunofluorescence experiments strongly indicated that YIPF3 is synthesized in the ER as a N-glycosylated form (40 kDa), is then O-glycosylated in the Golgi apparatus to become a lower mobility form (46 kDa) and finally becomes a higher mobility form cleaved at its C-terminal luminal domain (36 kDa). YIPF3 and YIPF4 form a complex in the Golgi apparatus, and this was suggested to be important for their proper localization and function. The knockdown of YIPF3 or YIPF4 in HeLa cells induced fragmentation of the Golgi apparatus, suggesting their involvement in the maintenance of the Golgi structure.