Background: Elevated levels of the inflammatory cytokine TNF-alpha are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-alpha and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-alpha or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation. Results: In this study, we analyzed the impact of TNF-alpha or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-alpha and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-alpha on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-alpha on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-alpha and IGF1 on miRNA abundance. Conclusions: The inhibitory effects of TNF-alpha or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-alpha or IGF1. This study indicates that miRNAs are mediators of the inhibitory effect of TNF-alpha on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-alpha by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-alpha or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets.