WNT5A activates noncanonical Wnt signaling pathways and has critical functions in early development, differentiation, and tissue homeostasis. Two major WNT5A protein isoforms, which in this study we term WNT5A-L(A) and WNT5A-S(B), have been identified that differ by 18 AA at their amino terminus. Functional differences between the isoforms have been indicated in studies utilizing cancer cell lines but the activities of the isoforms in normal cells and during differentiation have not been explored. We examined the WNT5A isoforms in the normal osteoblast cell line hFOB1.19. WNT5A-L(A) and WNT5A-S(B) transcripts increased from Days 3 to 21 of differentiation but WNT5A-S(B) showed a greater fold-change. In undifferentiated cells, there are 2-fold more WNT5A-L(A) than WNT5A-S(B) transcripts. Total intracellular WNT5A protein increased up to 3-fold during differentiation. siRNA knockdown of total WNT5A leads to a decrease in the expression of the differentiation markers, osteocalcin and RUNX2. Conditioned medium containing the isoform proteins [CM-L(A) and CM-S(B)] was used to analyze the effects of the isoforms on β-catenin and noncanonical signaling, proliferation, gene expression, and alkaline phosphatase (ALP) activity. Treatment with both CM-L(A) and CM-S(B) reduced β-catenin signaling. CM-L(A) but not CM-S(B) significantly increased the proliferation of nondifferentiated hFOB1.19 cells. CM-L(A) enhanced osteocalcin transcripts over 2-fold in differentiating cells, whereas CM-S(B) had no effect. Analysis of differentiating cells up to Day 21 revealed no significant effect of treatment with CM-L(A) or CM-S(B) on ALP activity or osteocalcin gene expression. pJNK levels were unaffected in proliferating cells by treatment with neither isoform. pPKC increased slightly in CM-L(A)treated cells at 15 min but by 2 h pPKC levels were less than the control. CM-S(B)had a more robust effect on pPKC levels that continued up to 2 h. Together these This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.