PLIN1, a perilipin family member, is an integral membrane protein that determines lipid droplet (LD) heterogeneity in differentiating adipocytes. Unlike class II LD proteins, PLIN1 contains an unconventional integral membrane segment (iMS) that mediates its insertion into the endoplasmic reticulum (ER) and facilitates high-affinity binding to LDs. This iMS excludes class II PLINs from PLIN1 LDs, leading to the formation of distinct LD populations. PLIN1 can move between the ER and LDs, but this movement is a gated process. When the iMS is removed, PLIN1 becomes a soluble, cytoplasmic LD protein with reduced LD affinity and switches its LD class specificity. In contrast, moving the iMS to PLIN2 leads to ER insertion and the formation of a separate LD class. These findings highlight the role of organelle targeting and lipid affinity differences in LD heterogeneity. PLIN1's high-affinity binding to LDs ensures its exclusion from the second LD class formed upon fatty acid uptake, while class II PLINs, with lower affinity, bind to the second LD class. The study reveals that PLIN1's unique membrane integration and high-affinity LD binding are critical for establishing LD heterogeneity in adipocytes. This understanding may inform therapeutic strategies for metabolic diseases such as fatty liver disease, type 2 diabetes, and obesity.PLIN1, a perilipin family member, is an integral membrane protein that determines lipid droplet (LD) heterogeneity in differentiating adipocytes. Unlike class II LD proteins, PLIN1 contains an unconventional integral membrane segment (iMS) that mediates its insertion into the endoplasmic reticulum (ER) and facilitates high-affinity binding to LDs. This iMS excludes class II PLINs from PLIN1 LDs, leading to the formation of distinct LD populations. PLIN1 can move between the ER and LDs, but this movement is a gated process. When the iMS is removed, PLIN1 becomes a soluble, cytoplasmic LD protein with reduced LD affinity and switches its LD class specificity. In contrast, moving the iMS to PLIN2 leads to ER insertion and the formation of a separate LD class. These findings highlight the role of organelle targeting and lipid affinity differences in LD heterogeneity. PLIN1's high-affinity binding to LDs ensures its exclusion from the second LD class formed upon fatty acid uptake, while class II PLINs, with lower affinity, bind to the second LD class. The study reveals that PLIN1's unique membrane integration and high-affinity LD binding are critical for establishing LD heterogeneity in adipocytes. This understanding may inform therapeutic strategies for metabolic diseases such as fatty liver disease, type 2 diabetes, and obesity.