Cotton is not only a natural fiber crop, but also an important economic crop in China. The cultivation of cotton with longer, stronger, and finer fiber has been the goal of cotton breeders. Germin-like proteins (GLPs) is a group of protein family with cupin domain, which mainly acts as an enzyme. However, the function of GLPs involved in regulating cotton fiber development is rarely understood. Here, we identified a gene named GbGLP2 from the G. barbadense cDNA library and elucidated its function negatively regulating fiber elongation that resulted in shorter fiber. The transcripts of GbGLP2 were mainly abundant in the elongating fiber of 10 days post-anthesis (DPA). GbGLP2 shared 99.8% similarity with GhGLP1. RNAi silencing of the homolog GhGLP1 in G. hirsutum accession YZ-1 produced longer fiber relative to the control. Enhancing the expression of GbGLP2 significantly reduced fiber length and strength. Through RNA-seq analysis between RNAi and WT lines, 566 genes differentially expressed were identified, of which 113 genes were downregulated and 453 genes were upregulated in the RNAi lines. Surprisingly, most of the downregulated genes participated in secondary cell wall biosynthesis, including cellulose synthase genes (CESA4, CESA7, and CESA8), hemicellulose biosynthesis genes (IRX9, COBL4) and transcription factors (MYB46, MYB86). Simultaneously, the transcriptome comparison between 30 cotton varieties with much longer fiber and 30 varieties with much shorter fiber revealed that the above downregulated genes were markedly decreased in long fiber. Taken together, these results suggest that knockdown of GhGLP1 promotes fiber elongation via delaying secondary cell wall thickening stage.