Long non-coding RNAs (lncRNAs) are emerging as a critical regulator in controlling the expression level of genes involved in cell differentiation and development. Primordial follicle activation (PFA) is the first step for follicle maturation, and excessive PFA results in premature ovarian insufficiency (POI). However, the correlation between lncRNA and cell differentiation was largely unknown, especially during PFA. In this study, we observed the expression level of lncRNA was more specific than protein-coding genes in both follicles and granulosa cells, suggesting lncRNA might play a crucial role in follicle development. Hence, a systematical framework was needed to infer the functions of lncRNAs during PFA. Additionally, an increasing number of studies indicate that the subpathway is more precise in reflecting biological processes than the entire pathway. Given the complex expression patterns of lncRNA target genes, target genes were further clustered based on their expression similarity and classification performance to reveal the activated/inhibited gene modules, which intuitively illustrated the diversity of lncRNA regulation. Moreover, the knockdown ofSBF2-AS1in the A549 cell line andZFAS1in the SK-Hep1 cell line further validated the function ofSBF2-AS1in regulating the Hippo signaling subpathway andZFAS1in the cell cycle subpathway. Overall, our findings demonstrated the importance of subpathway analysis in uncovering the functions of lncRNAs during PFA, and paved new avenues for future lncRNA-associated research.