1,25-dihydroxyvitamin D3 (vitamin D3) induces differentiation of HL-60 human myeloid leukemia cells; however, the signaling mechanism governing these effects is not fully clear. Here, we show that vitamin D3 induced functional differentiation by Akt through Raf/MEK/ERK MAPK signaling. Vitamin D3 downregulated Akt, weakened Akt-Raf1 interaction, and subsequently activated the Raf/MEK/ERK MAPK pathway. Pharmacological inhibition of MEK/ERK crippled differentiation in response to vitamin D3. Ectopic overexpression of Akt inhibited MAPK signaling, downregulated cyclin-dependent kinase (CDK) inhibitors p21(Wip1/Cip1) and p27(Kip1) and blunted differentiation in response to vitamin D3 while knockdown of Akt by RNA interference gave reverse effects. Furthermore, knockdown of the CDK inhibitors by siRNA crippled the recruitment of retinoblastoma protein (Rb) from the Raf1-Rb complex and Rb hypophosphorylation, and abolished differentiation in response to vitamin D3. Vitamin D3-induced MAPK signaling mediated upregulation of the CDK inhibitors and Rb, disassociation of Raf1 and Rb, and dephosphorylation of Rb, resulting in Rb binding to transcription factor E2F1 and subsequent differentiation. Finally, knockdown of Rb by siRNA prevented vitamin D3-induced differentiation. Mutating Rb at Ser795 evokes its association with E2F1, indicating the critical role of Rb Ser795 in regulating cell differentiation. Taken together, our data suggest that vitamin D3-triggered differentiation of human myeloid leukemia cells depends on downregulation of Akt, which dissociates from Raf1 and activates MAPK signaling leading to CDK inhibitor upregulation, Raf1 disassociation from Rb, and Rb upregulation and hypophosphorylation coupled to E2F1 binding.