Background: Trastuzumab (Tmab) has contributed to improving the prognosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. However, some cases did not respond or were resistant to Tmab. The combination of lapatinib with capecitabine (LC) is an effective treatment option for Tmab-refractory HER2-positive metastatic breast cancer. The aim of this study was to investigate the correlation between several biomarkers (PTEN expression, PIK3CA mutation, quantitative measures of HER2, p95HER2, and HER3) and treatment outcomes using LC in accordance with the estrogen receptor (ER) status. Methods: Eighty patients with HER2 positive breast cancer were enrolled in the prospective clinical trial (KBC-SG 1107) from December 2011 to March 2014. Total HER2 (H2T), p95HER2 (p95), and total HER3 (H3T) expression were quantified in formalin-fixed paraffin embedded samples using the VeraTag assays. The PTEN and p95 expression were detected by immunohistochemistry and PIK3CA mutation by direct sequencing. The median age was 58.0 years and the ER- and progesterone receptor (PgR)-positive rates were 55.0% and 33.8%, respectively. Results: The response rate to LC was 30% (CR: 1 case; PR: 23 cases), the clinical benefit rate was 51.3% and the median progression-free survival (PFS) was 174.5 days. ER negativity significantly correlated with a higher H2T and p95 expression. A lower H2T and PIK3CA mutation are often seen in non-responders (both: p = 0.087). The number of recurrence sites correlated with PFS but a high p95 expression correlated with a longer PFS in the higher H2T subset (especially in ER positive cases). PTEN, PIK3CA and HER3 did not correlate with the PFS and OS prognoses. OS after LC significantly correlated with the number of recurrence sites but not with the p95 expression. Conclusions: These findings suggest that LC therapy is effective in Tmab-refractory HER2 positive breast cancer. Moreover, the H2T and p95 expressions differed in ER status and a high p95 expression correlated with a longer PFS but not with OS. However, further studies are needed to confirm these findings.