Type 2 diabetes and skeletal muscle insulin resistance has been linked to accumulation of the intramyocellular lipid-intermediate diacylglycerol (DAG). However, recent animal and human studies have questioned such an association. Given that DAG appears in different stereoisomers and has different reactivity in vitro, we investigated if the described function of DAGs as mediators of lipid-induced insulin resistance was depending on the different DAG-isomers. We measured insulin stimulated glucose uptake in hormone sensitive lipase (HSL) knock out (KO) mice after treadmill exercise to stimulate accumulation of DAGs in skeletal muscle. We found that despite an increased DAG content in muscle after exercise in HSL KO mice, the HSL KO mice showed a higher insulin stimulated glucose uptake post exercise compared to wildtype. Further analysis of the chemical structure and cellular localization of DAG in skeletal muscle revealed that HSL KO mice accumulated sn-1,3 DAG and not sn-1,2 DAG. Accordingly, these results highlight the importance of taking the chemical structure and cellular localization of DAG into account when evaluating the role of DAG in lipid induced insulin resistance in skeletal muscle and that accumulation of sn-1,3 DAG originating from lipolysis does not inhibit insulin stimulated glucose uptake.