Intracellular signals involved in the maturation and function of osteoclasts are poorly understood. Here, we demonstrate that osteoclasts express multiple regulatory subunits of class I(A) phosphatidylinositol 3-kinase (PI3-K) although the expression of the full-length form of p85alpha is most abundant. In vivo, deficiency of p85alpha results in a significantly greater number of trabeculae and significantly lower spacing between trabeculae as well as increased bone mass in both males and females compared to their sex-matched wild-type controls. Consistently, p85alpha(-/-) osteoclast progenitors show impaired growth and differentiation, which is associated with reduced activation of Akt and mitogen-activated protein kinase extracellular signal-regulated kinase 1 (Erk1)/Erk2 in vitro. Furthermore, a significant reduction in the ability of p85alpha(-/-) osteoclasts to adhere to as well as to migrate via integrin alphavbeta3 was observed, which was associated with reduced bone resorption. Microarray as well as quantitative real-time PCR analysis of p85alpha(-/-) osteoclasts revealed a significant reduction in the expression of several genes associated with the maturation and migration of osteoclasts, including microphathalmia-associated transcription factor, tartrate-resistant acid phosphatase, cathepsin K, and beta3 integrin. Restoring the expression of the full-length form of p85alpha but not the version with a deletion of the Src homology-3 domain restored the maturation of p85alpha(-/-) osteoclasts to wild-type levels. These results highlight the importance of the full-length version of the p85alpha subunit of class I(A) PI3-K in controlling multiple aspects of osteoclast functions.