Pathological cardiac hypertrophy is a major contributor to heart failure (HF), resulting in high mortality rates worldwide; therefore, identifying key molecules in pathological cardiac hypertrophy is of critical importance for preventing or reversing HF. Tripartite motif 38 (Trim38) is an E3 ubiquitin ligase that serves a pivotal role in various diseases. The present study aimed to elucidate the regulatory role of Trim38 in pressure overload-induced pathological cardiac hypertrophy and to explore its underlying molecular mechanisms. The expression of Trim38 was decreased in hypertrophic heart tissues from a murine model of transverse aortic constriction (TAC) and in neonatal rat cardiomyocytes (NRCMs) treated with phenylephrine (PE). Furthermore, Trim38 knockout (Trim38-KO) aggravated cardiac hypertrophy after TAC, and Trim38 knockdown in cardiomyocytes increased cell cross section area, and upregulated the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) following treatment with PE. Ubiquitinomics analysis revealed that the MAPK signaling pathway was regulated by Trim38. Furthermore, western blotting confirmed that Trim38-KO activated TAK1 and JNK/P38. By contrast, Trim38 overexpression in NRCMs suppressed the JNK/P38 signaling pathway and inhibited the phosphorylation of TAK1. Furthermore, Trim38 knockdown resulted in a marked enhancement of TAK1 phosphorylation, concomitant with an augmentation of cardiomyocyte area and a significant upregulation of the hypertrophic biomarkers ANP and BNP. By contrast, infection with an adenovirus containing dominant-negative TAK1 inhibited TAK1 activity, which attenuated Trim38 knockdown-induced cardiomyocyte hypertrophy, confirming that TAK1 is a key molecule involved in the protective effects of Trim38 on cardiomyocytes. In conclusion, to the best of our knowledge, the present study is the first to reveal that Trim38 confers protection against pathological cardiac hypertrophy by inhibiting the TAK1/JNK/P38 signaling pathway; therefore, Trim38 may be a promising target for treating cardiac hypertrophy.