Neuronatin (Nnat) is selectively expressed in the neonatal brain and is involved in neuronal differentiation during brain development. However, Nnat also appears to be abundantly expressed in adipose tissue, and is conspicuously elevated in the adipose tissue of obese Zucker diabetic fatty rats compared with control lean Zucker lean control rats shown in our previous report. Here, we examined the expression of Nnat in adipose tissue and demonstrated that the ectopic expression of Nnat mediated by retroviral infection or stable transfection of 3T3-L1 pre-adipocytes stimulated differentiation into mature adipocytes with early induction of adipogenic transcription factors. Moreover, in 3T3-L1 cells overexpressing Nnat, increased intracellular free calcium levels and enhanced phosphorylation of cAMP-response element-binding protein (CREB) were observed, which appears to potentiate CCAAT/enhancer-binding protein (C/EBP)β, C/EBPδ, and C/EBPα transcriptional activities. Collectively, the data indicate that Nnat enhances CREB phosphorylation through increasing intracellular free calcium levels, which potentiates expression of adipogenic transcription factors resulting in heightened adipocyte differentiation. These findings contribute to a greater fundamental understanding of obesity, a clinically important risk factor in numerous diseases.