Protein kinases and protein phosphatases play an important role in the regulation of gene expression. In this study, influence of protein kinases inhibitors W7, trifluoperazine, staurosporine, genistein, and protein phosphatase inhibitor okadaic acid on adventitious shoot regeneration from calli of eastern white pine (Pinus strobus L.) has been studied. Our results demonstrated that W7 at 200 µM, trifluoperazine at 200 µM, staurosporine at 1.5 µM, genistein at 200 µM, and okadaic acid at 1.5 µM significantly decreased the frequency of adventitious shoot formation and average number of shoots per g fresh weight calli, respectively, compared with the control. Activity analysis of phosphoglucoisomerase (PGI) and SDS-PAGE analysis of total protein extracts suggested that the general protein synthesis was not affected in calli cultured on the medium with W7, trifluoperazine, staurosporine, genistein, and okadaic acid. These results demonstrated that protein kinase and phosphatase activities were associated with adventitious shoot regeneration. These findings could be useful for studying mechanisms of adventitious shoot organogenesis in eastern white pine and may be valuable to understand plant development.