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(1) Background: Adipogenesis is an important issue in human health and livestock meat quality that has received widespread attention and extensive study. However, alternative splicing events may generate multiple isoforms with different functions. This will lead to known knowledge being far more complex than before. (2) Methods: We studied the effects of two different TUSC5 isoforms (TUSC5A and TUSC5B) in cattle on adipogenesis by constructing over-expression cell models and RNA-sequencing methods. (3) Results: We discovered that over-expression of TUSC5A promotes the process of adipogenesis while over-expression of TUSC5B suppresses it. Eight important genes (PPARG, ACC1, FASN, SCD1, LPL, FABP4, GPDH, and GLUT4) during adipogenesis were significantly promoted (student's t-test, p < 0.05) by TUSC5A and suppressed by TUSC5B both before and after cell differentiation. By performing a comprehensive analysis using a RNA-seq strategy, we found that both up-regulated differentially expressed genes (DEGs, |log2FoldChange| ≥ 1, p ≤ 0.05) of TUSC5A and down-regulated DEGs of TUSC5B were significantly enriched in the adipogenesis related GO terms, and the PPAR signaling pathway may play important role in those differences. (4) Conclusions: Our study proved that over-expression of two TUSC5 isoforms would regulate adipogenesis in the opposite direction. It is important to understand the function of the TUSC5 gene correctly. [ABSTRACT FROM AUTHOR]