The primary cilium, a microtubule-based organelle that protrudes from the surface of most eukaryotic cell types, serves as a signaling hub, playing a vital role in development and maintaining tissue homeostasis. Given the ever-increasing significance of primary cilia in both health and disease, discovering new factors involved in ciliogenesis will enhance our comprehension of the role of this organelle. The macrophage migration inhibitory factor (MIF) has been acknowledged as a secreted cytokine involved in the pathogenesis of various human diseases. MIF is expressed and released ubiquitously by numerous cell types and tissues, and unlike other cytokines, intracellular MIF exhibits unique functional characteristics. In a recent study, it was reported that MIF is a significant contributor to cilia biogenesis and serves as a novel transcriptional regulator in homeostasis. Utilizing immunofluorescent staining and three-dimensional structured illumination microscopy (3D-SIM), which allows the visualization of cellular components with high specificity, the authors demonstrated that MIF is localized and forms a ring-like structure at the proximal end of the centrioles, where it influences cilia biogenesis by affecting the recruitment and accumulation of basal body proteins, including TTBK2, CP110, and CEP290. Here, we describe protocols and optimization tips on how to use standard labeling techniques with 3D-SIM to investigate the role of MIF in primary cilia in both human- and murine-derived ciliated cells. The utilization of 3D-SIM alongside standard staining techniques, while concurrently identifying MIF within the three-dimensional centrosomal structure, presents intriguing new opportunities for MIF in molecular cell biology.