Skeletal muscle expresses at least three p38 MAPKs (α, β, γ). However, no studies have examined the potential regulation of glucose uptake by p38γ, the isoform predominantly expressed in skeletal muscle and highly regulated by exercise. L6 myotubes were transfected with empty vector (pCAGGS), activating MKK6 (MKK6CA), or p38γ-specific siRNA. MKK6CA-transfected cells had higher rates of basal 2-deoxy-d-[³H]glucose (2-DG) uptake (P < 0.05) but lower rates of 2,4-dinitrophenol (DNP)-stimulated glucose uptake, an uncoupler of oxidative phosphorylation that operates through an insulin-independent mechanism (P < 0.05). These effects were reversed when MKK6CA cells were cotransfected with p38γ-specific siRNA. To determine whether the p38γ isoform is involved in the regulation of contraction-stimulated glucose uptake in adult skeletal muscle, the tibialis anterior muscles of mice were injected with pCAGGS or wild-type p38γ (p38γWT) followed by intramuscular electroporation. Basal and contraction-stimulated 2-DG uptake in vivo was determined 14 days later. Overexpression of p38γWT resulted in higher basal rates of glucose uptake compared with pCAGGS (P < 0.05). Muscles overexpressing p38γWT showed a trend for lower in situ contraction-mediated glucose uptake (P = 0.08) and significantly lower total GLUT4 levels (P < 0.05). These data suggest that p38γ increases basal glucose uptake and decreases DNP- and contraction-stimulated glucose uptake, partially by affecting levels of glucose transporter expression in skeletal muscle. These findings are consistent with the hypothesis that activation of stress kinases such as p38 are negative regulators of stimulated glucose uptake in peripheral tissues.